Modified peptide nucleic acid compositions

ABSTRACT

The present disclosure relates to compounds useful for the detection or modulation of target nucleic acids, including DNA and RNA. The present disclosure further relates to methods for treatment of trinucleotide repeat disorders, which can include administration of oligonucleotide analogues that can bind pathogenic nucleotide repeats in DNA or RNA.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.17/218,145, filed Mar. 30, 2021, which claims the benefit of U.S.Provisional Application No. 63/002,326, filed Mar. 30, 2020, each ofwhich is hereby incorporated by reference in its entirety.

BACKGROUND

Huntington's disease (HD) is a neurodegenerative genetic disease causedby a mutation in the huntingtin (HTT) gene, which is thought to beimportant for many essential cell activities. Those with HD experience aloss of neurons in the brain over time mostly due to the toxicaccumulation of faulty HTT protein in their cells. Current treatmentoptions work to lessen individual movement and psychiatric symptoms, butthere is no treatment for HD that can slow or stop the progression ofthe disease.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

In some embodiments, the present disclosure provides a compoundcomprising a chain, wherein the chain comprises a series of atomsconcatenated to form the chain, wherein a plurality of the atoms thatare concatenated to form the chain are each independently substitutedwith a substituent that bears a guanidino group, wherein the chain has apattern of one atom that is independently substituted with a substituentthat bears a guanidino group, followed by five consecutive atoms thatare not substituted by a substituent that bears a guanidino group,followed by a second atom that is independently substituted with asubstituent that bears a guanidino group, followed by another fiveconsecutive atoms that are not substituted by a substituent that bears aguanidino group, followed by a third atom that is independentlysubstituted with a substituent that bears a guanidino group, wherein afirst end of the chain or a second end of the chain is substituted witha peptide.

In some embodiments, the present disclosure provides a compoundcomprising a peptide nucleic acid sequence and a cell permeabilizinggroup attached to the peptide nucleic acid sequence, wherein if aradiolabeled analogue of the compound is subjected to an assay, whereinthe assay comprises:

-   -   (a) a first component, wherein the first component comprises:        -   (i) administering a 5 mg/kg intravenous bolus dose of the            radiolabeled analogue to a caudal vein of a monkey, wherein            the monkey is a male Cynomolgus monkey;        -   (ii) euthanizing the monkey 4 hours after the administering;        -   (iii) after (ii), freezing the monkey in a mixture of hexane            and solid carbon dioxide for at least two hours to provide a            frozen carcass;        -   (iv) embedding the frozen carcass, left lateral side            uppermost, in 2% w/v aqueous sodium carboxymethylcellulose            to provide an embedded carcass;        -   (v) sectioning the embedded carcass into 40 μm sagittal            whole body sections with a cryomacrotome;        -   (vi) mounting the 40 μm sagittal whole body sections on            pressure sensitive tape;        -   (vii) after (vi), dehydrating the whole body sections in the            cryomacrotome at about −20° C. for about 60 hours;        -   (viii) after (vii), placing the whole body sections against            an image plate sensitive to carbon-14 for no longer than            four days;        -   (ix) after (viii), scanning the image plate with a phosphor            imager system; and        -   (x) after (ix), determining a concentration of the            radiolabeled analogue in brain tissue of the whole body            sections; and    -   (b) a second component, wherein the second component is        analogous to the first component except that the second        component uses another monkey that is euthanized 168 hours after        the administering,    -   wherein the radiolabeled analogue comprises an N-terminus that        is substituted with a ¹⁴C-enriched glycine residue, and the        radiolabeled analogue consists of a structure that differs from        the compound solely in that the compound lacks the ¹⁴C-enriched        glycine residue of the radiolabeled analogue,        then in the assay, the concentration of the radiolabeled        analogue in brain tissue determined in the second component is        equivalent to at least about 80% of the concentration of the        radiolabeled analogue in brain tissue determined in the first        component.

In some embodiments, the present disclosure provides a compoundcomprising a peptide nucleic acid sequence, wherein the peptide nucleicacid sequence comprises: (i) a series of peptide nucleic acid residueshaving a repeating triad of nucleobase side chains; and (ii) a cellpermeabilizing group attached to the series of peptide nucleic acidresidues, wherein if the compound is subjected to an assay, and theassay comprises:

-   -   (a) administering by intracerebroventricular administration a        dose amount of about 0.1 mg/kg to about 2 mg/kg of the compound        to mice;    -   (b) euthanizing the mice at a time point between about 1 hour        and 28 days post intracerebroventricular administration;    -   (c) collecting brain tissues from the mice after the        euthanizing; and    -   (d) using liquid chromatography-tandem mass spectrometry to        determine concentrations of the brain tissues in the mice,        then in the assay, a mean maximum brain concentration is        observed in the mice at a time to maximum brain concentration of        about 1 hour to about 50 hours post administration and the mean        maximum brain concentration of the mice is observed to be about        3000 ng/mL to about 22000 ng/mL.

In some embodiments, the present disclosure provides a compoundcomprising a peptide nucleic acid sequence, wherein the peptide nucleicacid sequence comprises: (i) a series of peptide nucleic acid residueshaving a repeating triad of nucleobase side chains; and (ii) a cellpermeabilizing group attached to the series of peptide nucleic acidresidues, wherein if the compound is subjected to an assay, and theassay comprises:

-   -   (a) administering by intracerebroventricular administration a        dose amount of about 0.1 mg/kg to about 1.5 mg/kg of the        compound to mice;    -   (b) collecting blood samples from cava veins of the mice at a        time point between about 1 hour and 28 days post        intracerebroventricular administration;    -   (c) after the collecting the blood samples, euthanizing the mice        at a time point between about 1 hour and 28 days post        intracerebroventricular administration;    -   (d) after the euthanizing, collecting brain, intestine, liver,        lung, kidney, or muscle tissues from the mice; and    -   (e) using liquid chromatography-tandem mass spectrometry to        determine concentrations of the compound in the brain,        intestine, liver, lung, kidney, or muscle tissues that were        collected; and    -   (f) using the liquid chromatography-tandem mass spectrometry to        determine concentrations of the compound in plasma from the        blood samples that were collected from the mice,        then, in the assay, the compound is observed to accumulate in        the brain of the mice for at most about a month after the        administering and the compound is not observed at a detectable        level during the month in the plasma, intestine, liver, lung,        kidney, or muscle of the mice.

In some embodiments, the present disclosure provides a compoundcomprising a peptide nucleic acid sequence, wherein the peptide nucleicacid sequence comprises: (i) a series of peptide nucleic acid residueshaving a repeating triad of nucleobase side chains; and (ii) a cellpermeabilizing group attached to the series of peptide nucleic acidresidues, wherein if the compound is subjected to a plasma proteinbinding assay, and the plasma protein binding assay comprises:

-   -   (a) performing a human component of the plasma protein binding        assay, wherein the human component of the plasma protein binding        assay comprises (1) spiking single aliquots of human plasma with        a 10 mg/mL of a first solution of the compound to obtain at        least a second solution of the compound with concentrations of        about 1 μg/mL to about 50 μg/mL; (2) using ultracentrifugation        on the at least the second solution of the compound to separate        a mixture comprising the compounds that are bound to plasma        proteins; (3) using liquid chromatography-tandem mass        spectrometry to determine a plasma protein binding percentage in        the human plasma;    -   (b) performing a mouse component of the plasma protein binding        assay, wherein the mouse component of the plasma protein binding        assay differs from the human component of the plasma protein        binding assay only in that mouse plasma is used instead of the        human plasma;    -   (c) performing a dog component of the plasma protein binding        assay, wherein the dog component of the plasma protein binding        assay differs from the human component of the plasma protein        binding assay only in that dog plasma is used instead of the        human plasma;    -   (d) performing a minipig component of the plasma protein binding        assay, wherein the minipig component of the plasma protein        binding assay differs from the human component of the plasma        protein binding assay only in that minipig plasma is used        instead of the human plasma;    -   (e) performing a sheep component of the plasma protein binding        assay, wherein the sheep component of the plasma protein binding        assay differs from the human component of the plasma protein        binding assay only in that sheep plasma is used instead of the        human plasma; and    -   (f) performing a monkey component of the plasma protein binding        assay, wherein the monkey component of the plasma protein        binding assay differs from the human component of the plasma        protein binding assay only in that monkey plasma is used instead        of the human plasma,        then in the plasma protein binding assay, the plasma protein        binding percentage is at least about 85% in the human, mouse,        dog, minipig, sheep, or monkey.

In some embodiments, the present disclosure provides a compound havingthe formula (I):

-   -   wherein:        -   each B is independently a nucleobase;        -   each R¹ is independently a side chain of a natural amino            acid, a guanidino(C₁-C₄)alkyl, or hydrogen;        -   each R² is independently a side chain of a natural amino            acid, a guanidino(C₁-C₄)alkyl, or hydrogen;        -   R⁵ is a sequence comprising at least one alpha amino acid            residue, beta amino acid residue, gamma amino acid residue,            or a combination thereof; hydrogen; or a water solubilizing            group;        -   n is an integer from 3-30; and        -   G is OH, NH₂, or

-   -   -   wherein:            -   R³ is hydrogen or an amino(C₁-C₄)alkyl;            -   R⁴ is a sequence comprising at least one alpha amino                acid residue, beta amino acid residue, gamma amino acid                residue, or a combination thereof; or hydrogen; and            -   m is 0 or 1;                wherein the compound comprises at least one                guanine-cytosine-thymine sequence;                or a pharmaceutically-acceptable salt thereof.

In some embodiments, the present disclosure provides a method oftreating a condition in a subject, the method comprising administeringto the subject a therapeutically-effective amount of any compound of thedisclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates cumulative excretion of radioactivity following asingle intravenous bolus of [¹⁴C]-Compound 1 to a male cynomolgusmonkeys at 5 mg/kg (Animal 103).

FIG. 2 illustrates individual blood and plasma profiles of totalradioactivity following a single intravenous bolus of [¹⁴C]-Compound 1to a male cynomolgus monkeys at 5 mg/kg in Animal 101 (4 hours postdose, Panel A), Animal 102 (12 hours post dose, Panel B), Animal 103 (24hours post-dose, Panel C) and Animal 103 (168 hours post-dose, Panel D).

FIG. 3 illustrates concentrations of total radioactivity inrepresentative organs and tissues at various times following a singleintravenous bolus of [¹⁴C]-Compound 1 to male cynomolgus monkeys at 5mg/kg.

FIG. 4 illustrates concentrations of total radioactivity inrepresentative organs and tissues at various times following a singleintravenous bolus of [¹⁴C]-Compound 1 to male cynomolgus monkeys at 5mg/kg.

FIG. 5 illustrates tissue to blood ratios at various times following asingle intravenous bolus of [¹⁴C]-Compound 1 to male cynomolgus monkeysat 5 mg/kg.

FIG. 6 illustrates tissue to blood ratios at various times following asingle intravenous bolus of [¹⁴C]-Compound 1 to male cynomolgus monkeysat 5 mg/kg.

FIG. 7A and FIG. 7B are autoradiographs that depict representativetissue distribution of total radioactivity in parasagittal sections ofAnimal 101 following a single intravenous 5 mg/kg bolus of[¹⁴C]-Compound 1. Any grey signal above background indicates presence ofcompound in tissues.

FIG. 8A and FIG. 8B are autoradiographs that depict representativetissue distribution of total radioactivity in sagittal sections ofAnimal 101 following a single intravenous 5 mg/kg bolus of[¹⁴C]-Compound 1. Any grey signal above background indicates presence ofcompound in tissues.

FIG. 9A and FIG. 9B are autoradiographs that depict representativetissue distribution of total radioactivity in sagittal sections ofAnimal 101 following a single intravenous 5 mg/kg bolus of[¹⁴C]-Compound 1. Any grey signal above background indicates presence ofcompound in tissues.

FIG. 10A and FIG. 10B are autoradiographs that depict representativetissue distribution of total radioactivity in sagittal sections ofAnimal 101 following a single intravenous 5 mg/kg bolus of[¹⁴C]-Compound 1. Any grey signal above background indicates presence ofcompound in tissues.

FIG. 11 is an autoradiograph that depicts representative tissuedistribution of total radioactivity in a mid-sagittal section of Animal101 following a single intravenous 5 mg/kg bolus of [¹⁴C]-Compound 1.Any grey signal above background indicates presence of compound intissues.

FIG. 12A and FIG. 12B are autoradiographs that depict representativetissue distribution of total radioactivity in sagittal sections ofAnimal 101 (4 hours post dose), Animal 102 (12 hours post dose), Animal103 (7 days post dose) following a single intravenous 5 mg/kg bolus of[¹⁴C]-Compound 1. Any grey signal above background indicates presence ofcompound in tissues.

FIG. 13 are autoradiographs that depict representative cranial tissuedistribution of total radioactivity in selected sagittal sections ofAnimal 101 (4 hours post dose), Animal 102 (12 hours post dose), Animal103 (7 days post dose) following a single intravenous 5 mg/kg bolus of[¹⁴C]-Compound 1. Any grey signal above background indicates presence ofcompound in tissues.

FIG. 14 are autoradiographs that depict representative pelvic-areatissue distribution of total radioactivity in selected sagittal sectionsof Animal 101 (4 hours post dose), Animal 102 (12 hours post dose),Animal 103 (7 days post dose) following a single intravenous 5 mg/kgbolus of [¹⁴C]-Compound 1. Any grey signal above background indicatespresence of compound in tissues.

FIG. 15A illustrates a mean brain concentration vs time profile ofCompound 1 in C57BL6J mice administered doses of 0.3 mg/kg, FIG. 15Billustrates mean brain concentration vs time profile of Compound 1 inmice administered doses of 0.6 mg/kg, and FIG. 15C illustrates meanbrain concentration vs time profile of Compound 1 in C57BL6J miceadministered doses of 1 mg/kg.

FIG. 16A illustrates mean spleen concentration vs time profile ofCompound 1 in C57BL6J mice administered doses of 0.3 mg/kg, FIG. 16Billustrates mean spleen concentration vs time profile of Compound 1 inmice administered doses of 0.6 mg/kg, and FIG. 16 C illustrates meanspleen concentration vs time profile of Compound 1 in C57BL6J miceadministered doses of 1 mg/kg.

FIG. 17 illustrates mean heart concentration vs time profile of Compound1 in C57BL6J mice administered doses of 0.3 mg/kg.

FIG. 18A illustrates mean brain, spleen, and heart concentrations vstime profile of Compound 1 in C57BL6J mice administered doses of 0.3mg/kg, FIG. 18B illustrates mean brain, spleen, and heart concentrationsvs time profile of Compound 1 in C57BL6J mice administered doses of 0.6mg/kg, FIG. 18C illustrates mean brain, spleen, and heart concentrationsvs time profile of Compound 1 in C57BL6J mice administered doses of 1mg/kg.

FIG. 19A shows the relative expression of wtHTT (darker bars) and mHTT(lighter bars) by GM09197 cells after treatment with Compound 2 at 1 or5 μM.

FIG. 19B shows the relative expression of wtHTT (darker bars) and mHTT(lighter bars) by GM09197 cells after treatment with Compound 3 at 1 or5 μM.

FIG. 19C shows the relative expression of wtHTT (darker bars) and mHTT(lighter bars, not visible) by GM09197 cells after treatment withCompound 4 at 1 or 5 μM. The mHTT bars are not visible as mHTT was belowthe limit of detection.

FIG. 20A shows the results of a cytotoxicity assay evaluating thepercent of dead GM09197 cells present after mock-treatment (PBS,control) or treatment with Compound 2 at 1 or 5 μM.

FIG. 20B shows the results of a cytotoxicity assay evaluating thepercent of dead GM09197 cells present after mock-treatment (PBS,control) or treatment with Compound 3 at 1 or 5 μM.

FIG. 20C shows the results of a cytotoxicity assay evaluating thepercent of dead GM09197 cells present after mock-treatment (PBS,control) or treatment with Compound 4 at 1 or 5 μM.

FIG. 21A depicts the structural formula of Compound 1. FIG. 21B depictsthe structural formula of [¹⁴C]Compound 1.

FIG. 22A depicts the structural formula of Compound 2. FIG. 22B depictsthe structural formula of Compound 4.

FIG. 23 depicts the structural formula of Compound 3.

DETAILED DESCRIPTION

Huntington's disease (HD) is a genetic disease associated with anabnormally long CAG repeat expansion in the huntingtin gene (HTT), whichcodes for the huntingtin protein (HTT). HTT genes that contain repeatlengths beyond a certain threshold produce mutant huntingtin protein(mHTT), which can induce pathological changes in the central nervoussystem. The risk, penetrance, and age of disease onset can be correlatedwith length of the HTT repeat expansion. Repeat counts of less than 27CAG triads are associated with normal phenotype, while repeat countsfrom 27 to 35 generally confer normal phenotype but enhanced risk ofdisease in offspring. 36 to 39 repeats are associated with incomplete orreduced penetrance, with disease symptoms manifesting later in adultlife, if at all. Repeat lengths greater than 40 confer full penetrance,while repeat counts of greater than 60 produce disease that can manifestas early as childhood.

Compounds of the Disclosure.

The present disclosure relates to compounds useful for the detection ormodulation of target nucleic acids, including DNA and RNA. The presentdisclosure further relates to methods for treatment of trinucleotiderepeat disorders, which can include administration of oligonucleotideanalogues that can bind pathogenic nucleotide repeats in DNA or RNA. Insome embodiments, compounds of the disclosure bind CAG repeats withinthe mutant mHTT transcript present in Huntington's disease, therebymodulating expression of mHTT protein.

In some embodiments, the disclosure provides a compound comprising achain. The chain can comprise a series of atoms concatenated to form thechain. A plurality of the atoms that are concatenated to form the chaincan be independently substituted with a substituent that bears a polargroup, which can be, for example, a guanidino group. The chain can havea pattern of one atom that is independently substituted with asubstituent that bears a polar group, followed by five consecutive atomsthat are not substituted by a substituent that bears a polar group,followed by a second atom that is independently substituted with asubstituent that bears a polar group, followed by another fiveconsecutive atoms that are not substituted by a substituent that bears apolar group, followed by a third atom that is independently substitutedwith a substituent that bears a polar group.

In some embodiments, the chain has a pattern of one atom that isindependently substituted with a substituent that bears a polar group(e.g., guanidino group), followed by seventeen consecutive atoms thatare not substituted by a substituent that bears a polar group, followedby a second atom that is independently substituted with a substituentthat bears a polar group, followed by another seventeen consecutiveatoms that are not substituted by a substituent that bears a polargroup, followed by a third atom that is independently substituted with asubstituent that bears a polar group. In some embodiments, the thirdatom that is independently substituted with a substituent that bears apolar group (e.g., guanidino group) is followed by eleven consecutiveatoms that are not substituted by a substituent that bears a polargroup, followed by a fourth atom that is independently substituted witha substituent that bears a polar group. In some embodiments, the fourthatom that is independently substituted with a substituent that bears apolar group is followed by another eleven consecutive atoms that are notsubstituted by a substituent that bears a polar group. In someembodiments, the first atom, the second atom, and the third atom areeach gamma carbons of a peptide nucleic acid oligomer. In someembodiments, the first atom, the second atom, and the third atom areeach alpha carbons of a peptide nucleic acid oligomer.

In some embodiments, the chain can have a pattern of one atom that isindependently substituted with a substituent that bears a polar group(e.g., guanidino group), followed by eleven consecutive atoms that arenot substituted by a substituent that bears a polar group, followed by asecond atom that is independently substituted with a substituent thatbears a polar group, followed by another eleven consecutive atoms thatare not substituted by a substituent that bears a polar group, followedby a third atom that is independently substituted with a substituentthat bears a polar group.

Suitable polar groups can include groups that bear a formal charge atphysiological pH, such as a guanidino group. In some embodiments, eachsubstituent that bears a guanidino group is independentlyguanidinoalkyl. In some embodiments, each substituent that bears aguanidino group is independently guanidino(C₁-C₄)alkyl. In someembodiments, each substituent that bears a guanidino group is2-guanidino-eth-1-yl. In some embodiments, each substituent that bears aguanidino group is 3-guanidino-prop-1-yl. In some embodiments, eachsubstituent that bears a guanidino group is 4-guanidino-but-1-yl.

Compounds of the disclosure can comprise nucleobases or nucleobaseanalogs. In some embodiments, the pattern can further comprise one atomthat is independently substituted with a substituent that bears a firstnucleobase, followed by five consecutive atoms that are not substitutedby a substituent that bears a nucleobase, followed by a second atom thatis independently substituted with a substituent that bears a secondnucleobase, followed by another five consecutive atoms that are notsubstituted by a substituent that bears a nucleobase, followed by athird atom that is independently substituted with a substituent thatbears a third nucleobase.

In some embodiments, the substituent that bears the first nucleobase,the substituent that bears the second nucleobase, and the substituentthat bears the third nucleobase are each independently purinylacyl,purinylalkylene, pyrimidinylacyl, or pyrimidinylalkylene. In someembodiments, the substituent that bears the first nucleobase, thesubstituent that bears the second nucleobase, and the substituent thatbears the third nucleobase are each independently guaninylacyl,adeninylacyl, cytosinylacyl, thyminylacyl, or uracilylacyl. In someembodiments, the first nucleobase, the second nucleobase, and the thirdnucleobase form a sequence that is CTG, TGC, or GCT.

In some embodiments, a compound of the disclosure can be resistant todegradation by enzymes (e.g. nucleases or proteases). In someembodiments, the compound can be stable in a subject. In someembodiments, a compound of the disclosure can be water-soluble. In someembodiments, the compound can be endocytosed by a cell comprising atarget sequence of the compound. In some embodiments, the compound isendocytosed, pinocytosed, phagocytosed in a cell that does not containthe target sequence. In some embodiments, the compound is transcytosedacross the endothelia lining of the cerebral vasculature, or“blood-brain barrier.”

Peptide Nucleic Acids.

The present disclosure peptide nucleic acid analogs andpharmaceutically-acceptable salts thereof. In some embodiments, thecompound comprises a peptide nucleic acid domain. Peptide nucleic acidsare oligonucleotide analogues that comprise a chain of repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds, where the glycylnitrogen of one or more units is functionalized with an alkylene or acylgroup bearing a nucleobase. Peptide nucleic acids can optionallycomprise substitution on the N-(2-aminoethyl)-glycine backbone, forexample:

where substituents R^(α), R^(β), R^(γ), are alpha, beta, and gammasubstituents, respectively. In some embodiments, the peptide nucleicacid chain is substituted with a polar group, such as a group thatcomprises a guanidino moiety. The polar group can be bound to the alphaor gamma position of at least one peptide nucleic acid subunit.

A compound of the disclosure (e.g., a peptide nucleic acid) can comprisea chain of atoms with termini that are substituted or unsubstituted. Forexample, a first end of the chain and a second end of the chain can beeach independently unsubstituted or substituted with an amino acid. Forexample, a first end of the chain and a second end of the chain can beeach independently unsubstituted or substituted with a peptide. In someembodiments, the compound is a peptide nucleic acid oligomer, whereinthe first end of the chain is an N-terminus of the peptide nucleic acidoligomer, and the second end of the chain is a C-terminus of the peptidenucleic acid oligomer. In some embodiments, the C-terminus of thepeptide nucleic acid oligomer is bound by a peptide bond to a peptide,which can be for example, a sequence comprising alpha amino acidresidues, beta amino acid residues, gamma amino acid residues, or acombination thereof. In some embodiments, the C-terminus of the peptidenucleic acid oligomer is bound by a peptide bond to amidated lysine. Insome embodiments, the C-terminus of the peptide nucleic acid oligomer isbound by a peptide bond to amidated beta-lysine.

In some embodiments, the C-terminus or N-terminus of the peptide nucleicacid is substituted with a cell-permeabilizing group. In someembodiments, the cell permeabilizing group is a polypeptide comprising 3to 8 lysine residues. In some embodiments, the polypeptide is linked tothe peptide nucleic acid via an amide bond. In some embodiments, thepolypeptide is linked to the peptide nucleic acid via a peptide bond, adisulfide bond, or a linker comprising two penicillamine residues boundby a disulfide bond.

In some embodiments, the compound can comprise from 4 to 10 guanidinogroups. In some embodiments, the compound can comprise from 6 to 8guanidino groups. In some embodiments, the compound can comprise from 7to 9 guanidino groups. In some embodiments, the compound can comprise 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more guanidinogroups. Each of the three guanidino groups can be independently bound tothe peptide nucleic acid chain. In some embodiments, the group thatcomprises a guanidino moiety is a 4-guanidino-but-1-yl group, a3-guanidino-prop-1-yl group, or a 2-guanidino-eth-1-yl group. In someembodiments, a sequence of the peptide nucleic acid domain comprises(GCT)_(n), wherein n is 1-10. In some embodiments, a sequence of thepeptide nucleic acid domain is domain (GCT)₆G. In In some embodiments, asequence of the peptide nucleic acid domain is GCTGCT. In someembodiments, a sequence of the peptide nucleic acid domain is CTGCTG.

In some embodiments, a compound of the disclosure can comprise a moietythat improves cell-permeability of the compound relative to a moleculewithout the moiety that is otherwise identical to the compound. Forexample, a compound of the disclosure can reach an intracellular targetwithin the cytoplasm or nucleus.

In some embodiments, the disclosure provides a compound comprising apharmacophore region attached to a multiply-positively charged region,wherein:

-   -   a) the pharmacophore region comprises a number of peptide        nucleic acid residues, wherein the number of peptide nucleic        acid residues is at least 7 and is not a multiple of 3;    -   b) each peptide nucleic acid residue of the pharmacophore region        independently comprises a backbone part and a side chain part        attached to the backbone part;    -   c) none of the backbone parts of the peptide nucleic acid        residues of the pharmacophore region bears a positive formal        charge at neutral pH;    -   d) each side chain part independently bears a nucleobase;    -   e) the nucleobases of each of the side chain parts collectively        form a sequence;    -   f) the sequence is complementary to a native, human nucleic acid        sequence associated with Huntington's Disease;    -   g) the sequence comprises a subsequence that is        cytosine-thymine-guanine-cytosine-thymine-guanine; and    -   h) the multiply-positively charged region comprises at least six        consecutive building blocks, wherein each of the consecutive        building blocks independently comprises a side chain that        carries a positive formal charge at neutral pH.

In some embodiments, the present disclosure provides a compoundrepresented by the structure of formula (I):

wherein

-   -   each B is independently a nucleobase;    -   each R¹ is independently a side chain of a natural amino acid, a        guanidino(C₁-C₄)alkyl, or hydrogen;    -   each R² is independently a side chain of a natural amino acid, a        guanidino(C₁-C₄)alkyl, or hydrogen;    -   R⁵ is a sequence comprising at least one alpha amino acid        residue, beta amino acid residue, gamma amino acid residue, or a        combination thereof; hydrogen; or a water solubilizing group;    -   n is an integer from 3-30;    -   G is OH, NH₂, or

-   -   wherein    -   R³ is hydrogen or an amino(C₁-C₄)alkyl;    -   R⁴ is a sequence comprising at least one alpha amino acid        residue, beta amino acid residue, gamma amino acid residue, or a        combination thereof; or hydrogen; and    -   m is 0 or 1;    -   wherein the compound comprises at least one        guanine-cytosine-thymine sequence; or a        pharmaceutically-acceptable salt thereof or a radiolabeled        derivative thereof.

In some embodiments, each nucleobase B is independently guanine,thymine, or cytosine. In some embodiments, B is guanine. In someembodiments, B is thymine. In some embodiments, B is cytosine. In someembodiments, B is adenine. In some embodiments, B is uracil. In someembodiments, each nucleobase B is an analog of a naturally occurringnucleobase.

In some embodiments, at least one R¹ is guanidino(C₁-C₄)alkyl. In someembodiments, more than one R¹ is guanidino(C₁-C₄)alkyl. In someembodiments, at least one every other R¹ is guanidino(C₁-C₄)alkyl. Insome embodiments, at least one every third R¹ is guanidino(C₁-C₄)alkyl.In some embodiments, at least one every second or third R¹ isguanidino(C₁-C₄)alkyl. In some embodiments, each R¹ isguanidino(C₁-C₄)alkyl. In some embodiments, at least one R¹ is4-guanidinobut-1-yl. In some embodiments, at least one R¹ is3-guanidinoprop-1-yl. In some embodiments at least one R¹ is2-guanidino-eth-1-yl. In some embodiments, at least one R¹ is hydrogen.In some embodiments, each R¹ is hydrogen.

In some embodiments, at least one R² is guanidino(C₁-C₄)alkyl. In someembodiments, more than one R² is guanidino(C₁-C₄)alkyl. In someembodiments, at least one every other R² is guanidino(C₁-C₄)alkyl. Insome embodiments, at least one every third R² is guanidino(C₁-C₄)alkyl.In some embodiments, at least one every second or third R² isguanidino(C₁-C₄)alkyl. In some embodiments, each R² isguanidino(C₁-C₄)alkyl. In some embodiments, at least one R² is4-guanidinobut-1-yl. In some embodiments, at least one R² is3-guanidinoprop-1-yl. In some embodiments at least one R² is2-guanidino-eth-1-yl. In some embodiments, at least one R² is hydrogen.In some embodiments, each R² is hydrogen.

In some embodiments, G is

In some embodiments, R⁵ is the water-solubilizing group. In someembodiments, the water-solubilizing group is a multiply-positivelycharged region that comprises at least six consecutive building blocks.In some embodiments, each of the consecutive building blocksindependently comprises a side chain that carries a positive formalcharge at neutral pH.

In some embodiments, R³ is hydrogen. In some embodiments, R³ is anamino(C₁-C₄)alkyl. In some embodiments, R³ is 4-aminobut-1-yl. In someembodiments, R³ is 3-aminoprop-1-yl. In some embodiments, m is 0. Insome embodiments, m is 1.

In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ is asequence comprising at least one alpha amino acid residue. In someembodiments, R⁴ is a sequence comprising at least one beta amino acidresidue. In some embodiments, R⁴ is a sequence comprising at least onegamma amino acid residue. In some embodiments, R⁴ is

-   -   wherein        -   p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and        -   each R⁶ is independently hydrogen or an amino(C₁-C₄)alkyl.

In some embodiments, R⁴ is

In some embodiments, p is 3, 4, 5, 6, 7, or 8. In some embodiments, p is3. In some embodiments, p is 4. In some embodiments, p is 5. In someembodiments, p is 6. In some embodiments, p is 7. In some embodiments, pis 8.

In some embodiments, R⁵ is hydrogen.

In some embodiments, R⁵ is

-   -   wherein        -   each R⁷ is independently a side chain of a natural amino            acid; and        -   q is 0 or 1

In some embodiments, R⁷ is a side chain of an alpha amino acid. In someembodiments, R⁷ is a side chain of an beta amino acid. In someembodiments, R⁷ is a side chain of a gamma amino acid. In someembodiments, q is 0. In some embodiments, q is 1.

In some embodiments, R⁵ is

In some embodiments, R⁵ is

In some embodiments, the water solubilizing group comprises a structurethat has multiple formal charges at physiological pH. In someembodiments, R⁵ is the multiple formal charges are positive charges.

In some embodiments, when G is OH or NH₂, at least one of R¹ and R² is aside chain of a natural amino acid or a guanidino(C₁-C₄)alkyl, and R⁵ isnot hydrogen.

In some embodiments, when R¹ and R² are both hydrogen, G is

In some embodiments, when R¹ and R² are both hydrogen, G is

and R⁵ is the sequence comprising at least one alpha amino acid residue.

In some embodiments, the compound has the formula:

In some embodiments, n is 6. In some embodiments, at least one R¹ is4-guanidinobut-1-yl. In some embodiments, each R¹ is4-guanidinobut-1-yl. In some embodiments, at least one R² is hydrogen.In some embodiments, each R² is hydrogen. In some embodiments, R³ is4-aminobut-1-yl. In some embodiments, R³ is 3-aminoprop-1-yl. In someembodiments, R⁴ is hydrogen. In some embodiments, R⁵ is hydrogen. Insome embodiments, R⁵ is

In some embodiments, the compound has the formula:

-   -   wherein        -   each B^(1a), B^(2a) and B^(3a) is independently cytosine,            guanine, or thymine; and        -   n¹ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, n¹ is 2. In some embodiments, the compoundcomprises the sequence (CTG)₂. In some embodiments, at least one R¹ is4-guanidinobut-1-yl. In some embodiments, each R¹ is4-guanidinobut-1-yl. In some embodiments, at least one R¹ is hydrogen.In some embodiments, R³ is 4-aminobut-1-yl. In some embodiments, R³ is3-aminoprop-1-yl.

In some embodiments, the compound has the formula:

In some embodiments, B^(1a) is cytosine, B^(2a) is thymine and B^(3a) isguanine. In some embodiments, n¹ is 2.

In some embodiments, the compound has the formula:

In some embodiments, the compound has the formula:

In some embodiments, n is 6. In some embodiments, the compound comprisesthe sequence (CTG)₂. In some embodiments, at least one R¹ is4-guanidinobut-1-yl. In some embodiments, each R¹ is4-guanidinobut-1-yl. In some embodiments, at least one R¹ is hydrogen.In some embodiments, R³ is 4-aminobut-1-yl. In some embodiments, R³ is3-aminoprop-1-yl.

In some embodiments, the compound has the formula:

-   -   wherein        -   each B^(1b), B^(2b) and B^(3b) is independently cytosine,            guanine or thymine; and        -   n² is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, n² is 2. In some embodiments, R¹ is4-guanidinobut-1-yl. In some embodiments, R³ is 4-aminobut-1-yl. In someembodiments, R³ is 3-aminoprop-1-yl.

In some embodiments, the compound has the formula:

In some embodiments, B^(1b) is cytosine, B^(2b) is thymine and B^(3b) isguanine. In some embodiments, n² is 2.

In some embodiments, the compound has the formula:

In some embodiments, the compound has the formula:

In some embodiments, the compound has the formula:

-   -   wherein        -   p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

each R⁶ is independently hydrogen or an amino(C₁-C₄)alkyl.

In some embodiments, n is 10. In some embodiments, n is 11. In someembodiments, n is 12. In some embodiments, n is 13. In some embodiments,n is 14. In some embodiments, n is 15. In some embodiments, n is 16. Insome embodiments, n is 17. In some embodiments, n is 18. In someembodiments, n is 19. In some embodiments, n is 20.

In some embodiments, at least one R¹ is hydrogen. In some embodiments, nis greater than 1, and every other R¹ is hydrogen. In some embodiments,at least one R¹ is 4-guanidinobut-1-yl. In some embodiments, n isgreater than 1, and every other R¹ is 4-guanidinobut-1-yl.

In some embodiments, at least one R² is hydrogen. In some embodiments,each R² is hydrogen. In some embodiments, at least one R² is4-guanidinobut-1-yl. In some embodiments, every other R² is4-guanidinobut-1-yl.

In some embodiments, R³ is 4-aminobut-1-yl. In some embodiments, R³ is3-aminoprop-1-yl.

In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ is

-   -   wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, p is 3. In some embodiments, p is 4. In someembodiments, p is 5. In some embodiments, p is 6. In some embodiments, pis 7. In some embodiments, p is 8.

In some embodiments, R⁵ is hydrogen. In some embodiments, R⁵ is

In some embodiments, R⁵ is

In some embodiments, R⁶ is 4-aminobut-1-yl. In some embodiments, R⁶ is3-aminoprop-1-yl.

In some embodiments, the compound has the formula:

-   -   wherein        -   R⁷ is a side chain of a natural amino acid.

In some embodiments, R⁷ is 4-aminobut-1-yl. In some embodiments, R⁷ is3-aminoprop-1-yl.

In some embodiments, the compound has the structure

-   -   wherein        -   each B^(1c), B^(2c), B^(3c) and B^(4c) is independently            cytosine, guanine or thymine; and        -   n³ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, B^(1c) is guanine. In some embodiments, B^(2c) iscytosine. In some embodiments, B^(3c) is thymine. In some embodiments,B^(4c) is guanine.

In some embodiments, n³ is 6.

In some embodiments, p is 7.

In some embodiments, R³ is 4-aminobut-1-yl. In some embodiments, R³ is3-aminoprop-1-yl.

In some embodiments, R⁶ is 4-aminobut-1-yl. In some embodiments, R⁶ is3-aminoprop-1-yl.

In some embodiments, R⁷ is 4-aminobut-1-yl. In some embodiments, R⁷ is3-aminoprop-1-yl.

In some embodiments, the compound comprises a sequence comprisingG(CTG)₆.

In some embodiments, the compound has the structure

In some embodiments, the compound has the structure

In some embodiments, the compound has the structure

-   -   wherein        -   R⁷ is a side chain of a natural amino acid.

In some embodiments, R⁷ is 4-aminobut-1-yl. In some embodiments, R⁷ is3-aminoprop-1-yl.

In some embodiments, the compound has the structure

-   -   wherein        -   X is

-   -   -   Y is

-   -   -   each B^(1d), B^(2d), B^(3d), B^(4d), B^(5d), B^(6d), B^(7d),            B^(8d), B^(9d), and B^(10d) is independently cytosine,            guanine or thymine;        -   n⁴ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and        -   n⁵ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, B^(1d) is guanine. In some embodiments, B^(2d) iscytosine. In some embodiments, B^(3d) is thymine. In some embodiments,B^(4d) is guanine. In some embodiments, B^(5d) is cytosine. In someembodiments, B^(6d) is thymine. In some embodiments, B^(7d) is guanine.In some embodiments, B^(8d) is cytosine. In some embodiments, B^(9d) isthymine. In some embodiments, B^(10d) is guanine.

In some embodiments, n⁴ is 3. In some embodiments, n⁵ is 2.

In some embodiments, R³ is 4-aminobut-1-yl. In some embodiments, atleast one R¹ is 4-guanidinobut-1-yl. In some embodiments, each R¹ is4-guanidinobut-1-yl.

In some embodiments, R³ is 4-aminobut-1-yl. In some embodiments, R³ is3-aminoprop-1-yl. In some embodiments,

In some embodiments, R⁷ is 4-aminobut-1-yl. In some embodiments, R⁷ is3-aminoprop-1-yl.

In some embodiments, the compound has the structure:

wherein

X¹ is

and

Y¹ is

In some embodiments, the compound has the structure:

wherein

X² is

and

Y² is

In some embodiments, the compound has the structure:

wherein

-   -   L is

-   -   M is

-   -   each B^(1e), B^(2e), B^(3e), B^(4e), B^(5e), B^(6e), B^(7e),        B^(8e), B^(9e) and B^(10e) is independently cytosine, guanine or        thymine;    -   n⁶ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   n⁷ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, B^(1e) is guanine. In some embodiments, B^(2e) iscytosine. In some embodiments, B^(3e) is thymine. In some embodiments,B^(4e) is guanine. In some embodiments, B^(5e) is cytosine. In someembodiments, B^(6e) is thymine. In some embodiments, B^(7e) is guanine.In some embodiments, B^(8e) is cytosine. In some embodiments, B^(9e) isthymine. In some embodiments, B^(10e) is guanine.

In some embodiments, n⁶ is 3. In some embodiments, n⁷ is 2.

In some embodiments, at least one R² is 3-guanidinoprop-1-yl. In someembodiments, each R² is 3-guanidinoprop-1-yl. In some embodiments, atleast one R² is 4-guanidinobut-1-yl. In some embodiments, each R² is4-guanidinobut-1-yl.

In some embodiments, R³ is 4-aminobut-1-yl. In some embodiments, R³ is3-aminoprop-1-yl.

In some embodiments, R⁷ is 4-aminobut-1-yl. In some embodiments, R⁷ is3-aminoprop-1-yl.

In some embodiments, the compound has the structure:

wherein

L¹ is

and

M¹ is

In some embodiments, the compound has the structure:

-   -   wherein    -   L² is

and

-   -   M² is

Chemical Groups.

Each chemical group disclosed herein may be unsubstituted orsubstituted. Non-limiting examples of optional substituents includehydroxyl groups, sulfhydryl groups, halogens, amino groups, nitrogroups, nitroso groups, cyano groups, azido groups, sulfoxide groups,sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehydegroups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups,halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups,aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups,heterocyclyl groups, acyl groups, hydrocarbyl groups, acyloxy groups,carbamate groups, amide groups, and ester groups.

Non-limiting examples of alkyl and alkylene groups include straight,branched, and cyclic alkyl and alkylene groups. An alkyl group can be,for example, a C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃,C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇,C₂₈, C₂₉, C₃₀, C₃₁, C₃₂, C₃₃, C₃₄, C₃₅, C₃₆, C₃₇, C₃₈, C₃₉, C₄₀, C₄₁,C₄₂, C₄₃, C₄₄, C₄₅, C₄₆, C₄₇, C₄₈, C₄₉, or C₅₀ group that is substitutedor unsubstituted. A (C₁-C₄) alkyl groups is an alkyl group comprisingbetween one and four carbon atoms.

Non-limiting examples of straight alkyl groups include methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

Branched alkyl groups include any straight alkyl group substituted withany number of alkyl groups. Non-limiting examples of branched alkylgroups include isopropyl, isobutyl, sec-butyl, and t-butyl.

Non-limiting examples of cyclic alkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptlyl, and cyclooctylgroups. Cyclic alkyl groups also include fused-, bridged-, andspiro-bicycles and higher fused-, bridged-, and spiro-systems. A cyclicalkyl group can be substituted with any number of straight, branched, orcyclic alkyl groups.

Non-limiting examples of alkenyl and alkenylene groups include straight,branched, and cyclic alkenyl groups. The olefin or olefins of an alkenylgroup can be, for example, E, Z, cis, trans, terminal, or exo-methylene.An alkenyl or alkenylene group can be, for example, a C₂, C₃, C₄, C₅,C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀,C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₁, C₃₂, C₃₃, C₃₄,C₃₅, C₃₆, C₃₇, C₃₈, C₃₉, C₄₀, C₄₁, C₄₂, C₄₃, C₄₄, C₄₅, C₄₆, C₄₇, C₄₈,C₄₉, or C₅₀ group that is substituted or unsubstituted.

Non-limiting examples of alkynyl or alkynylene groups include straight,branched, and cyclic alkynyl groups. The triple bond of an alkylnyl oralkynylene group can be internal or terminal. An alkylnyl or alkynylenegroup can be, for example, a C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅,C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₁, C₃₂, C₃₃, C₃₄, C₃₅, C₃₆, C₃₇, C₃₈, C₃₉,C₄₀, C₄₁, C₄₂, C₄₃, C₄₄, C₄₅, C₄₆, C₄₇, C₄₈, C₄₉, or C₅₀ group that issubstituted or unsubstituted.

A halo-alkyl group can be any alkyl group substituted with any number ofhalogen atoms, for example, fluorine, chlorine, bromine, and iodineatoms. A halo-alkenyl group can be any alkenyl group substituted withany number of halogen atoms. A halo-alkynyl group can be any alkynylgroup substituted with any number of halogen atoms.

An alkoxy group can be, for example, an oxygen atom substituted with anyalkyl, alkenyl, or alkynyl group. An ether or an ether group comprisesan alkoxy group. Non-limiting examples of alkoxy groups include methoxy,ethoxy, propoxy, isopropoxy, and isobutoxy.

An aryl group can be heterocyclic or non-heterocyclic. An aryl group canbe monocyclic or polycyclic. An aryl group can be substituted with anynumber of substituents described herein, for example, hydrocarbylgroups, alkyl groups, alkoxy groups, and halogen atoms. Non-limitingexamples of aryl groups include phenyl, toluyl, naphthyl, pyrrolyl,pyridyl, imidazolyl, thiophenyl, and furyl.

An aryloxy group can be, for example, an oxygen atom substituted withany aryl group, such as phenoxy.

An aralkyl group can be, for example, any alkyl group substituted withany aryl group, such as benzyl.

An arylalkoxy group can be, for example, an oxygen atom substituted withany aralkyl group, such as benzyloxy.

A heterocycle can be any ring containing a ring atom that is not carbon,for example, N, O, S, P, Si, B, or any other heteroatom. A heterocyclecan be substituted with any number of substituents, for example, alkylgroups and halogen atoms. A heterocycle can be aromatic (heteroaryl) ornon-aromatic. Non-limiting examples of heterocycles include pyrrole,pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine,imidazole, thiophene, furan, tetrahydrofuran, pyran, andtetrahydropyran.

An acyl group can be, for example, a carbonyl group substituted withhydrocarbyl, alkyl, hydrocarbyloxy, alkoxy, aryl, aryloxy, aralkyl,arylalkoxy, or a heterocycle. Non-limiting examples of acyl includeacetyl, benzoyl, benzyloxycarbonyl, phenoxycarbonyl, methoxycarbonyl,and ethoxycarbonyl.

An acyloxy group can be an oxygen atom substituted with an acyl group.An ester or an ester group comprises an acyloxy group. A non-limitingexample of an acyloxy group, or an ester group, is acetate.

A carbamate group can be an oxygen atom substituted with a carbamoylgroup, wherein the nitrogen atom of the carbamoyl group isunsubstituted, monosubstituted, or disubstituted with one or more ofhydrocarbyl, alkyl, aryl, heterocyclyl, or aralkyl. When the nitrogenatom is disubstituted, the two substituents together with the nitrogenatom can form a heterocycle.

A hydrocarbyl group can be any group consisting of carbon and hydrogenatoms, and can include alkyl groups, alkenyl groups, alkynyl groups, andaryl groups. A hydrocarbyl group can be, for example, a C₂, C₃, C₄, C₅,C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀,C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₁, C₃₂, C₃₃, C₃₄,C₃₅, C₃₆, C₃₇, C₃₈, C₃₉, C₄₀, C₄₁, C₄₂, C₄₃, C₄₄, C₄₅, C₄₆, C₄₇, C₄₈,C₄₉, or C₅₀ group.

A hydrocarbylcarbonyl group can be a carbonyl group substituted with ahydrocarbyl group, which can be, for example, benzoyl, acetyl,propanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl,decanoyl, undencanoyl, dodecanoyl, tridencanoyl, myristoyl,pentadecenoyl, palmitoyl, heptadecanoyl, stearoyl, nondecanoyl,arachidoyl, as well as acyl groups derived from saturated,monounsaturated, and polyunsaturated fatty acids, such as myristoleoyl,palmitoleoyl, sapienoyl, oleoyl, elaidoyl, vaccenoyl, linoleoyl,linoelaidoyl, α-linolenoyl, or arachidonoyl. A hydrocarbylcarbonyl groupcan be, for example, a C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂,C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆,C₂₇, C₂₈, C₂₉, C₃₀, C₃₁, C₃₂, C₃₃, C₃₄, C₃₅, C₃₆, C₃₇, C₃₈, C₃₉, C₄₀,C₄₁, C₄₂, C₄₃, C₄₄, C₄₅, C₄₆, C₄₇, C₄₈, C₄₉, or C₅₀ group.

An aminoalkylene group can be an alkyl group substituted with an aminogroup, such as, for example, aminomethyl, 2-aminoeth-1-yl,3-aminoprop-1-yl, 2-aminoprop-1-yl, 4-aminobut-1-yl, 3-aminobut-1-yl,2-aminobut-1-yl, 5-aminopen-1-yl, 4-aminopent-1-yl, 4-aminopent-1-yl,3-aminopent-1-yl, 2-aminopent-1-yl, a lysine side chain, or an ornithineside chain.

A guanidinoalkylene group can be an alkyl group substituted with aguanidino group, such as, for example, guanidinomethyl,2-guanidinoeth-1-yl, 3-guanidinoprop-1-yl, 2-guanidinoprop-1-yl,4-guanidinobut-1-yl, 3-guanidinobut-1-yl, 2-guanidinobut-1-yl,5-guanidinopent-1-yl, 4-guanidinopent-1-yl, 4-guanidinopent-1-yl,3-guanidinopent-1-yl, 2-guanidinopent-1-yl, an arginine side chain, or ahomoarginine side chain.

“Polypeptide”, “peptide” and their grammatical equivalents as usedherein refer to a polymer of amino acid residues. Polypeptides andproteins disclosed herein (including functional portions and functionalvariants thereof) can comprise synthetic amino acids in place of one ormore naturally-occurring amino acids. The disclosure contemplates bothL- and D-forms of amino acid residues.

A compound of the disclosure may be radiolabeled. One or more of theatoms of the compound of the disclosure may be substituted with aradioactive or non-radioactive isotope, for example of ²H, ³H, ¹¹C, ¹³C,¹⁴C, 13N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, or combinations thereof. In oneembodiment, at least one carbon of the compound of the disclosure may besubstituted with ¹⁴C.

Therapeutic Methods.

The present disclosure describes the use of a compound and methods totreat conditions or genetic disease, including trinucleotide repeatdisorders. The method can comprise administering to the subject atherapeutically-effective amount of a compound of the disclosure. Insome embodiments, the genetic disease is a polyglutamine (polyQ)disease. Polyglutamine diseases include trinucleotide repeat disordersinvolving genes that comprise an abnormally high number of CAG repeats.In some embodiments, the polyglutamine disease is SCAT (Spinocerebellarataxia Type 1), SCA2 (Spinocerebellar ataxia Type 2), SCA3(Spinocerebellar ataxia Type 3 or Machado-Joseph disease), SCA6(Spinocerebellar ataxia Type 6), SCAT (Spinocerebellar ataxia Type 7),SCA12 (Spinocerebellar ataxia Type 12), SCA17 (Spinocerebellar ataxiaType 17), DRPLA (Dentatorubropallidoluysian atrophy), SBMA (Spinal andbulbar muscular atrophy), or Huntington's disease.

In some embodiments, the condition is a neurological condition. In someembodiments, the neurological condition is Huntington's disease. In someembodiments, the condition is a central nervous system condition. Insome embodiments, the condition is associated with aging. In someembodiments, the condition is associated with cognitive impairment. Insome embodiments, the condition is associated with memory loss. In someembodiments, the condition is associated with deterioration of motorskills.

In some embodiments, the polyglutamine disease is Huntington's disease.Treatment can be administered on the basis of number of CAG repeats inthe HTT gene of a subject. For example, a subject administered acompound of the disclosure can comprise a HTT gene that contains morethan 27 CAG repeats. In some embodiments, the HTT gene of the subjectcontains at least 36 repeats, at least 40 repeats, at least 50 repeats,or at least 60 repeats. In some embodiments, the HTT gene of the subjectcontains from 27 to 36 repeats, from 27 to 36 repeats, from 27 to 40repeats, from 27 to 60 repeats, from 27 to 80 repeats, from 27 to 90repeats, from 36 to 40 repeats, from 36 to 60 repeats, from 36 to 80repeats, from 36 to 90 repeats, from 40 to 60 repeats, from 40 to 80repeats, from 40 to 90 repeats, or from 60 to 90 repeats.

In some embodiments, administration of a compound of the disclosure doesnot exhibit immunogenicity. In some embodiments, administration of acompound of the disclosure does not promote generation of neutralizingantibodies, complement factors, pro-inflammatory cytokines, or type 1interferons upon or after administration of the compound to a subject.In some embodiments, the compounds do not activate the TLR9 receptor andare not presented in MHCI or MHCII complexes to the immune system.

Compounds of the disclosure can be systemically administered to asubject in need thereof as a therapeutically-effective amount of acompound that binds to a repeat codon. The subject can comprise abloodstream, a brain, and a blood-brain-barrier. The compound that bindsto the repeat codon can enter the brain by passing from the bloodstreamthrough the blood-brain-barrier into the brain.

Modes of Administration

A compound of the disclosure or a composition comprising a compound ofthe disclosure (for example, a pharmaceutical composition) can beadministered to a subject in various forms and by various suitableroutes of administration.

A compound of the disclosure or a composition comprising a compound ofthe disclosure (for example, a pharmaceutical composition) can beadministered in a local manner, for example, via injection of thecompound directly into an organ, optionally in a depot or sustainedrelease formulation or implant. A compound of the disclosure or acomposition comprising a compound of the disclosure (for example, apharmaceutical composition) can be administered in a systemic manner.

In some embodiments, a compound of the disclosure or a compositioncomprising a compound of the disclosure (for example, a pharmaceuticalcomposition) is administered parenterally. Parenteral administration canbe, for example, by bolus injection or by gradual infusion or perfusionover time. Administration can also be by surgical deposition of a bolus,or positioning of a medical device.

In some embodiments, a compound of the disclosure or a compositioncomprising a compound of the disclosure (for example, a pharmaceuticalcomposition) is administered orally. In some embodiments, a compound ofthe disclosure or a composition comprising a compound of the disclosure(for example, a pharmaceutical composition) is administered by anintravenous, intratumoral, subcutaneous, intramuscular, intracerebral,intracerebroventricular, intra-articular, intraperitoneal, intracranial,intrathecal, intranasal, buccal, sublingual, oral, or rectaladministration route. In some embodiments, a compound of the disclosureor a composition comprising a compound of the disclosure (for example, apharmaceutical composition) is administered by intravenousadministration. In some embodiments, a compound of the disclosure or acomposition comprising a compound of the disclosure (for example, apharmaceutical composition) is administered by subcutaneousadministration. In some embodiments, a compound of the disclosure or acomposition comprising a compound of the disclosure (for example, apharmaceutical composition) is administered by intracerebroventricularadministration. In some embodiments, a compound of the disclosure or acomposition comprising a compound of the disclosure (for example, apharmaceutical composition) is administered by oral administration. Insome embodiments, a compound of the disclosure or a compositioncomprising a compound of the disclosure (for example, a pharmaceuticalcomposition) is administered by intrathecal administration.

Any aforementioned route of administration can be combined with anotherroute of administration. For example, a compound of the disclosure canbe delivered by a first route of administration, and one or moresubsequent maintenance doses of the compound can be delivered by thesame or a different route of administration. In some embodiments, acompound of the disclosure or a composition comprising a compound of thedisclosure (for example, a pharmaceutical composition) is administeredby intrathecal administration, and one or more subsequent maintenancedoses of the compound or the composition comprising the compound aredelivered by subcutaneous administration or intravenous administration.

Non-limiting examples of suitable modes and routes of administrationinclude oral, topical, parenteral, intravenous injection, intravenousinfusion, subcutaneous injection, subcutaneous infusion, intramuscularinjection, intramuscular infusion, intradermal injection, intradermalinfusion, intraperitoneal injection, intraperitoneal infusion,intracerebral injection, intracerebral infusion, subarachnoid injection,subarachnoid infusion, intraocular injection, intraspinal injection,intrasternal injection, ophthalmic administration, endothelialadministration, local administration, intranasal administration,intrapulmonary administration, rectal administration, intraarterialadministration, intrathecal administration, inhalation, intralesionaladministration, intradermal administration, epidural administration,absorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa), intracapsular administration,subcapsular administration, intracardiac administration, transtrachealadministration, subcuticular administration, subarachnoidadministration, subcapsular administration, intraspinal administration,and intrasternal administration.

A compound of the disclosure or a composition comprising a compound ofthe disclosure (for example, a pharmaceutical composition) can beadministered via a non-invasive method. Examples of non-invasive modesof administering can include using a needleless injection device, andtopical administration, for example, eye drops. Multiple administrationroutes can be employed for efficient delivery.

Depending on the intended mode of administration, the compositions canbe in the form of solid, semi solid or liquid dosage forms, such as, forexample, tablets, suppositories, pills, capsules, powders, liquids,suspensions, lotions, creams, or gels, for example, in unit dosage formsuitable for single administration of a precise dosage. The compositioncan be formulated into any suitable dosage form for administration, forexample, aqueous dispersions, liquids, gels, syrups, elixirs, slurries,and suspensions, for administration to a subject or a patient.

Solid compositions include, for example, powders, tablets, dispersiblegranules, capsules, and cachets. Liquid compositions include, forexample, solutions in which a compound is dissolved, emulsionscomprising a compound, or a solution containing liposomes, micelles, ornanoparticles comprising a compound as disclosed herein. Semi-solidcompositions include, for example, gels, suspensions and creams. Thecompositions can be in liquid solutions or suspensions, solid formssuitable for solution or suspension in a liquid prior to use, or asemulsions. These compositions can also contain minor amounts ofnontoxic, auxiliary substances, such as wetting or emulsifying agents,pH buffering agents, and other pharmaceutically-acceptable additives.

In some embodiments, the composition is formulated into solutions (forexample, for IV administration). In some cases, the pharmaceuticalcomposition is formulated as an infusion. In some cases, thepharmaceutical composition is formulated as an injection.

A compound of the disclosure or a composition comprising a compound ofthe disclosure (for example, a pharmaceutical composition) can beadministered in the form of a rapid release formulation, in the form ofan extended release formulation, or in the form of an intermediaterelease formulation. A rapid release form can provide an immediaterelease. An extended release formulation can provide a controlledrelease or a sustained delayed release.

A composition comprising a compound of the disclosure can be, forexample, an immediate release form or a controlled release formulation.An immediate release formulation can be formulated to allow thecompounds to act rapidly. Non-limiting examples of immediate releaseformulations include readily dissolvable formulations. A controlledrelease formulation can be a pharmaceutical formulation that has beenadapted such that release rates and release profiles of the active agentcan be matched to physiological and chronotherapeutic requirements, orhas been formulated to effect release of an active agent at a programmedrate. Non-limiting examples of controlled release formulations includegranules, delayed release granules, hydrogels (e.g., of synthetic ornatural origin), other gelling agents (e.g., gel-forming dietaryfibers), matrix-based formulations (e.g., formulations comprising apolymeric material having at least one active ingredient dispersedthrough), granules within a matrix, polymeric mixtures, and granularmasses.

In some embodiments, a controlled release formulation is a delayedrelease form. A delayed release form can be formulated to delay acompound's action for an extended period of time. A delayed release formcan be formulated to delay the release of an effective dose of one ormore compounds, for example, for about 4, about 8, about 12, about 16,or about 24 hours. A controlled release formulation can be a sustainedrelease form. A sustained release form can be formulated to sustain, forexample, the compound's action over an extended period of time. Asustained release form can be formulated to provide an effective dose ofany compound described herein (e.g., provide a physiologically-effectiveblood profile) over about 4, about 8, about 12, about 16, or about 24hours.

A pharmaceutical composition disclosed herein can be targeted to anysuitable tissue or cell type. Modes, routes, and compositions of thedisclosure can be suitable to target a compound of the disclosure to aparticular tissue, or a subset of tissues. Non-limiting examples oftissues that can be targeted include kidney (e.g., kidney cortex),joints, cartilage, liver, salivary glands, bone (e.g., bone surface),skin, lung, muscle, pancreas, hair follicles, large intestine mucosa,aortic wall, small intestine mucosa, adrenal gland, stomach mucosa,spleen, bone marrow, lymph nodes, thymus, brain, cerebellum, olfactorybulb, thalamus, caudate putamen, cerebral cortex, substantia nigra,lateral ventricle, choroid plexus, and combinations thereof

Dosing

Pharmaceutical compositions described herein can be in unit dosage formssuitable for single administration of precise dosages. In unit dosageform, the formulation is divided into unit doses containing appropriatequantities of one or more compound. The dosage (e.g.,therapeutically-effective amount) for a compound described herein can bein any amount necessary.

A compound described herein can be present in a composition or a unitdose in a range of from about 1 mg to about 2000 mg; from about 5 mg toabout 1000 mg, from about 10 mg to about 25 mg, from about 50 mg toabout 250 mg, from about 100 mg to about 200 mg, from about 1 mg toabout 50 mg, from about 50 mg to about 100 mg, from about 100 mg toabout 150 mg, from about 150 mg to about 200 mg, from about 200 mg toabout 250 mg, from about 250 mg to about 300 mg, from about 300 mg toabout 350 mg, from about 350 mg to about 400 mg, from about 400 mg toabout 450 mg, from about 450 mg to about 500 mg, from about 500 mg toabout 550 mg, from about 550 mg to about 600 mg, from about 600 mg toabout 650 mg, from about 650 mg to about 700 mg, from about 700 mg toabout 750 mg, from about 750 mg to about 800 mg, from about 800 mg toabout 850 mg, from about 850 mg to about 900 mg, from about 900 mg toabout 950 mg, or from about 950 mg to about 1000 mg.

A compound described herein can be present in a composition or a unitdose in a range of from about 1 μg to about 2000 μg; from about 5 μg toabout 1000 μg, from about 10 μg to about 25 μg, from about 50 μg toabout 250 μg, from about 100 μg to about 200 μg, from about 1 μg toabout 50 μg, from about 50 μg to about 100 μg, from about 100 μg toabout 150 μg, from about 150 μg to about 200 μg, from about 200 μg toabout 250 μg, from about 250 μg to about 300 μg, from about 300 μg toabout 350 μg, from about 350 μg to about 400 μg, from about 400 μg toabout 450 μg, from about 450 μg to about 500 μg, from about 500 μg toabout 550 μg, from about 550 μg to about 600 μg, from about 600 μg toabout 650 μg, from about 650 μg to about 700 μg, from about 700 μg toabout 750 μg, from about 750 μg to about 800 μg, from about 800 μg toabout 850 μg, from about 850 μg to about 900 μg, from about 900 μg toabout 950 μg, or from about 950 μg to about 1000 μg.

A compound described herein can be present in a composition or a unitdose in an amount of about 0.001 mg, about 0.002 mg, about 0.003 mg,about 0.004 mg, about 0.005 mg, about 0.006 mg, about 0.007 mg, about0.008 mg, about 0.009 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg,about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg,about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg,about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg,about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg,about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg,about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg,about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg,about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850mg, about 1900 mg, about 1950 mg, or about 2000 mg.

In some embodiments, a composition is present in a composition or a unitdose in an amount that is at least about 0.001 mg, at least about 0.002mg, at least about 0.003 mg, at least about 0.004 mg, at least about0.005 mg, at least about 0.006 mg, at least about 0.007 mg, at leastabout 0.008 mg, at least about 0.009 mg, at least about 0.01 mg, atleast about 0.02 mg, at least about 0.03 mg, at least about 0.04 mg, atleast about 0.05 mg, at least about 0.06 mg, at least about 0.07 mg, atleast about 0.08 mg, at least about 0.09 mg, at least about 0.1 mg, atleast about 0.2 mg, at least about 0.3 mg, at least about 0.4 mg, atleast about 0.5 mg, at least about 0.6 mg, at least about 0.7 mg, atleast about 0.8 mg, at least about 0.9 mg, at least about 1 mg, at leastabout 2 mg, at least about 3 mg, at least about 4 mg, at least about 5mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, atleast about 25 mg, at least about 30 mg, at least about 35 mg, at leastabout 40 mg, at least about 45 mg, at least about 50 mg, at least about55 mg, at least about 60 mg, at least about 65 mg, at least about 70 mg,at least about 75 mg, at least about 80 mg, at least about 85 mg, atleast about 90 mg, at least about 95 mg, at least about 100 mg, at leastabout 125 mg, at least about 150 mg, at least about 175 mg, at leastabout 200 mg, at least about 250 mg, at least about 300 mg, at leastabout 350 mg, at least about 400 mg, at least about 450 mg, at leastabout 500 mg, at least about 550 mg, at least about 600 mg, at leastabout 650 mg, at least about 700 mg, at least about 750 mg, at leastabout 800 mg, at least about 850 mg, at least about 900 mg, at leastabout 950 mg, at least about 1000 mg, at least about 1050 mg, at leastabout 1100 mg, at least about 1150 mg, at least about 1200 mg, at leastabout 1250 mg, at least about 1300 mg, at least about 1350 mg, at leastabout 1400 mg, at least about 1450 mg, at least about 1500 mg, at leastabout 1550 mg, at least about 1600 mg, at least about 1650 mg, at leastabout 1700 mg, at least about 1750 mg, at least about 1800 mg, at leastabout 1850 mg, at least about 1900 mg, at least about 1950 mg, or atleast about 2000 mg.

In some embodiments, a composition is present in a composition or a unitdose in an amount that is at most about 0.001 mg, at most about 0.002mg, at most about 0.003 mg, at most about 0.004 mg, at most about 0.005mg, at most about 0.006 mg, at most about 0.007 mg, at most about 0.008mg, at most about 0.009 mg, at most about 0.01 mg, at most about 0.02mg, at most about 0.03 mg, at most about 0.04 mg, at most about 0.05 mg,at most about 0.06 mg, at most about 0.07 mg, at most about 0.08 mg, atmost about 0.09 mg, at most about 0.1 mg, at most about 0.2 mg, at mostabout 0.3 mg, at most about 0.4 mg, at most about 0.5 mg, at most about0.6 mg, at most about 0.7 mg, at most about 0.8 mg, at most about 0.9mg, at most about 1 mg, at most about 2 mg, at most about 3 mg, at mostabout 4 mg, at most about 5 mg, at most about 10 mg, at most about 15mg, at most about 20 mg, at most about 25 mg, at most about 30 mg, atmost about 35 mg, at most about 40 mg, at most about 45 mg, at mostabout 50 mg, at most about 55 mg, at most about 60 mg, at most about 65mg, at most about 70 mg, at most about 75 mg, at most about 80 mg, atmost about 85 mg, at most about 90 mg, at most about 95 mg, at mostabout 100 mg, at most about 125 mg, at most about 150 mg, at most about175 mg, at most about 200 mg, at most about 250 mg, at most about 300mg, at most about 350 mg, at most about 400 mg, at most about 450 mg, atmost about 500 mg, at most about 550 mg, at most about 600 mg, at mostabout 650 mg, at most about 700 mg, at most about 750 mg, at most about800 mg, at most about 850 mg, at most about 900 mg, at most about 950mg, at most about 1000 mg, at most about 1050 mg, at most about 1100 mg,at most about 1150 mg, at most about 1200 mg, at most about 1250 mg, atmost about 1300 mg, at most about 1350 mg, at most about 1400 mg, atmost about 1450 mg, at most about 1500 mg, at most about 1550 mg, atmost about 1600 mg, at most about 1650 mg, at most about 1700 mg, atmost about 1750 mg, at most about 1800 mg, at most about 1850 mg, atmost about 1900 mg, at most about 1950 mg, or at most about 2000 mg.

In some embodiments, a dose (e.g., a unit dose) is about 0.001 mg/kg,about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg, about 0.005mg/kg, about 0.006 mg/kg, about 0.007 mg/kg, about 0.008 mg/kg, about0.009 mg/kg, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 2 mg/kg,about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg,about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg,about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 250mg/kg, about 300 mg/kg, about 350 mg/kg, about 400 mg/kg, about 450mg/kg, about 500 mg/kg, about 550 mg/kg, about 600 mg/kg, about 650mg/kg, about 700 mg/kg, about 750 mg/kg, about 800 mg/kg, about 850mg/kg, about 900 mg/kg, about 950 mg/kg, about 1000 mg/kg, about 1050mg/kg, about 1100 mg/kg, about 1150 mg/kg, about 1200 mg/kg, about 1250mg/kg, about 1300 mg/kg, about 1350 mg/kg, about 1400 mg/kg, about 1450mg/kg, about 1500 mg/kg, about 1550 mg/kg, about 1600 mg/kg, about 1650mg/kg, about 1700 mg/kg, about 1750 mg/kg, about 1800 mg/kg, about 1850mg/kg, about 1900 mg/kg, about 1950 mg/kg, or about 2000 mg/kg based onbody mass of a subject or a patient.

In some embodiments, a dose (e.g., a unit dose) is at least about 0.001mg/kg, at least about 0.002 mg/kg, at least about 0.003 mg/kg, at leastabout 0.004 mg/kg, at least about 0.005 mg/kg, at least about 0.006mg/kg, at least about 0.007 mg/kg, at least about 0.008 mg/kg, at leastabout 0.009 mg/kg, at least about 0.01 mg/kg, at least about 0.02 mg/kg,at least about 0.03 mg/kg, at least about 0.04 mg/kg, at least about0.05 mg/kg, at least about 0.06 mg/kg, at least about 0.07 mg/kg, atleast about 0.08 mg/kg, at least about 0.09 mg/kg, at least about 0.1mg/kg, at least about 0.2 mg/kg, at least about 0.3 mg/kg, at leastabout 0.4 mg/kg, at least about 0.5 mg/kg, at least about 0.6 mg/kg, atleast about 0.7 mg/kg, at least about 0.8 mg/kg, at least about 0.9mg/kg, at least about 1 mg/kg, at least about 2 mg/kg, at least about 3mg/kg, at least about 4 mg/kg, at least about 5 mg/kg, at least about 10mg/kg, at least about 15 mg/kg, at least about 20 mg/kg, at least about25 mg/kg, at least about 30 mg/kg, at least about 35 mg/kg, at leastabout 40 mg/kg, at least about 45 mg/kg, at least about 50 mg/kg, atleast about 55 mg/kg, at least about 60 mg/kg, at least about 65 mg/kg,at least about 70 mg/kg, at least about 75 mg/kg, at least about 80mg/kg, at least about 85 mg/kg, at least about 90 mg/kg, at least about95 mg/kg, at least about 100 mg/kg, at least about 125 mg/kg, at leastabout 150 mg/kg, at least about 175 mg/kg, at least about 200 mg/kg, atleast about 250 mg/kg, at least about 300 mg/kg, at least about 350mg/kg, at least about 400 mg/kg, at least about 450 mg/kg, at leastabout 500 mg/kg, at least about 550 mg/kg, at least about 600 mg/kg, atleast about 650 mg/kg, at least about 700 mg/kg, at least about 750mg/kg, at least about 800 mg/kg, at least about 850 mg/kg, at leastabout 900 mg/kg, at least about 950 mg/kg, at least about 1000 mg/kg, atleast about 1050 mg/kg, at least about 1100 mg/kg, at least about 1150mg/kg, at least about 1200 mg/kg, at least about 1250 mg/kg, at leastabout 1300 mg/kg, at least about 1350 mg/kg, at least about 1400 mg/kg,at least about 1450 mg/kg, at least about 1500 mg/kg, at least about1550 mg/kg, at least about 1600 mg/kg, at least about 1650 mg/kg, atleast about 1700 mg/kg, at least about 1750 mg/kg, at least about 1800mg/kg, at least about 1850 mg/kg, at least about 1900 mg/kg, at leastabout 1950 mg/kg, or at least about 2000 mg/kg based on body mass of asubject or a patient.

In some embodiments, a dose (e.g., a unit dose) is at most about 0.001mg/kg, at most about 0.002 mg/kg, at most about 0.003 mg/kg, at mostabout 0.004 mg/kg, at most about 0.005 mg/kg, at most about 0.006 mg/kg,at most about 0.007 mg/kg, at most about 0.008 mg/kg, at most about0.009 mg/kg, at most about 0.01 mg/kg, at most about 0.02 mg/kg, at mostabout 0.03 mg/kg, at most about 0.04 mg/kg, at most about 0.05 mg/kg, atmost about 0.06 mg/kg, at most about 0.07 mg/kg, at most about 0.08mg/kg, at most about 0.09 mg/kg, at most about 0.1 mg/kg, at most about0.2 mg/kg, at most about 0.3 mg/kg, at most about 0.4 mg/kg, at mostabout 0.5 mg/kg, at most about 0.6 mg/kg, at most about 0.7 mg/kg, atmost about 0.8 mg/kg, at most about 0.9 mg/kg, at most about 1 mg/kg, atmost about 2 mg/kg, at most about 3 mg/kg, at most about 4 mg/kg, atmost about 5 mg/kg, at most about 10 mg/kg, at most about 15 mg/kg, atmost about 20 mg/kg, at most about 25 mg/kg, at most about 30 mg/kg, atmost about 35 mg/kg, at most about 40 mg/kg, at most about 45 mg/kg, atmost about 50 mg/kg, at most about 55 mg/kg, at most about 60 mg/kg, atmost about 65 mg/kg, at most about 70 mg/kg, at most about 75 mg/kg, atmost about 80 mg/kg, at most about 85 mg/kg, at most about 90 mg/kg, atmost about 95 mg/kg, at most about 100 mg/kg, at most about 125 mg/kg,at most about 150 mg/kg, at most about 175 mg/kg, at most about 200mg/kg, at most about 250 mg/kg, at most about 300 mg/kg, at most about350 mg/kg, at most about 400 mg/kg, at most about 450 mg/kg, at mostabout 500 mg/kg, at most about 550 mg/kg, at most about 600 mg/kg, atmost about 650 mg/kg, at most about 700 mg/kg, at most about 750 mg/kg,at most about 800 mg/kg, at most about 850 mg/kg, at most about 900mg/kg, at most about 950 mg/kg, at most about 1000 mg/kg, at most about1050 mg/kg, at most about 1100 mg/kg, at most about 1150 mg/kg, at mostabout 1200 mg/kg, at most about 1250 mg/kg, at most about 1300 mg/kg, atmost about 1350 mg/kg, at most about 1400 mg/kg, at most about 1450mg/kg, at most about 1500 mg/kg, at most about 1550 mg/kg, at most about1600 mg/kg, at most about 1650 mg/kg, at most about 1700 mg/kg, at mostabout 1750 mg/kg, at most about 1800 mg/kg, at most about 1850 mg/kg, atmost about 1900 mg/kg, at most about 1950 mg/kg, or at most about 2000mg/kg based on body mass of a subject or a patient.

In some embodiments, a dose (e.g., a unit dose) is about 1 mg/kg toabout 2000 mg/kg; from about 5 mg/kg to about 1000 mg/kg, from about 10mg/kg to about 25 mg/kg, from about 50 mg/kg to about 250 mg/kg, fromabout 100 mg/kg to about 200 mg/kg, from about 1 mg/kg to about 50mg/kg, from about 50 mg/kg to about 100 mg/kg, from about 100 mg/kg toabout 150 mg/kg, from about 150 mg/kg to about 200 mg/kg, from about 200mg/kg to about 250 mg/kg, from about 250 mg/kg to about 300 mg/kg, fromabout 300 mg/kg to about 350 mg/kg, from about 350 mg/kg to about 400mg/kg, from about 400 mg/kg to about 450 mg/kg, from about 450 mg/kg toabout 500 mg/kg, from about 500 mg/kg to about 550 mg/kg, from about 550mg/kg to about 600 mg/kg, from about 600 mg/kg to about 650 mg/kg, fromabout 650 mg/kg to about 700 mg/kg, from about 700 mg/kg to about 750mg/kg, from about 750 mg/kg to about 800 mg/kg, from about 800 mg/kg toabout 850 mg/kg, from about 850 mg/kg to about 900 mg/kg, from about 900mg/kg to about 950 mg/kg, from about 950 mg/kg to about 1000 mg/kg,about 1 μg/kg to about 2000 μg/kg; from about 5 μg/kg to about 1000μg/kg, from about 10 μg/kg to about 25 μg/kg, from about 50 μg/kg toabout 250 μg/kg, from about 100 μg/kg to about 200 μg/kg, from about 1μg/kg to about 50 μg/kg, from about 50 μg/kg to about 100 μg/kg, fromabout 100 μg/kg to about 150 μg/kg, from about 150 μg/kg to about 200μg/kg, from about 200 μg/kg to about 250 μg/kg, from about 250 μg/kg toabout 300 μg/kg, from about 300 μg/kg to about 350 μg/kg, from about 350μg/kg to about 400 μg/kg, from about 400 μg/kg to about 450 μg/kg, fromabout 450 μg/kg to about 500 μg/kg, from about 500 μg/kg to about 550μg/kg, from about 550 μg/kg to about 600 μg/kg, from about 600 μg/kg toabout 650 μg/kg, from about 650 μg/kg to about 700 μg/kg, from about 700μg/kg to about 750 μg/kg, from about 750 μg/kg to about 800 μg/kg, fromabout 800 μg/kg to about 850 μg/kg, from about 850 μg/kg to about 900μg/kg, from about 900 μg/kg to about 950 μg/kg, or from about 950 μg/kgto about 1000 μg/kg based on body mass of a subject or a patient.

Pharmaceutical compositions and formulations described herein cancomprise, for example, a compound of the disclosure at any suitableconcentration. A formulation can comprise a composition of thedisclosure at a concentration of, for example, about 0.001 mg/mL, about0.002 mg/mL, about 0.003 mg/mL, about 0.004 mg/mL, about 0.005 mg/mL,about 0.006 mg/mL, about 0.007 mg/mL, about 0.008 mg/mL, about 0.009mg/mL, about 0.01 mg/mL, about 0.02 mg/mL, about 0.03 mg/mL, about 0.04mg/mL, about 0.05 mg/mL, about 0.06 mg/mL, about 0.07 mg/mL, about 0.08mg/mL, about 0.09 mg/mL, about 0.1 mg/mL, about 0.2 mg/mL, about 0.3mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1 mg/mL, about 2 mg/mL,about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 10 mg/mL, about 15mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL,about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL,about 85 mg/mL, about 90 mg/mL, about 95 mg/mL, about 100 mg/mL, about125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 250mg/mL, about 300 mg/mL, about 350 mg/mL, about 400 mg/mL, about 450mg/mL, about 500 mg/mL, about 550 mg/mL, about 600 mg/mL, about 650mg/mL, about 700 mg/mL, about 750 mg/mL, about 800 mg/mL, about 850mg/mL, about 900 mg/mL, about 950 mg/mL, about 1000 mg/mL, about 1050mg/mL, about 1100 mg/mL, about 1150 mg/mL, about 1200 mg/mL, about 1250mg/mL, about 1300 mg/mL, about 1350 mg/mL, about 1400 mg/mL, about 1450mg/mL, about 1500 mg/mL, about 1550 mg/mL, about 1600 mg/mL, about 1650mg/mL, about 1700 mg/mL, about 1750 mg/mL, about 1800 mg/mL, about 1850mg/mL, about 1900 mg/mL, about 1950 mg/mL, or about 2000 mg/mL.

In some embodiments, a formulation of the disclosure comprises acompound of the disclosure at a concentration of at least about 0.001mg/mL, at least about 0.002 mg/mL, at least about 0.003 mg/mL, at leastabout 0.004 mg/mL, at least about 0.005 mg/mL, at least about 0.006mg/mL, at least about 0.007 mg/mL, at least about 0.008 mg/mL, at leastabout 0.009 mg/mL, at least about 0.01 mg/mL, at least about 0.02 mg/mL,at least about 0.03 mg/mL, at least about 0.04 mg/mL, at least about0.05 mg/mL, at least about 0.06 mg/mL, at least about 0.07 mg/mL, atleast about 0.08 mg/mL, at least about 0.09 mg/mL, at least about 0.1mg/mL, at least about 0.2 mg/mL, at least about 0.3 mg/mL, at leastabout 0.4 mg/mL, at least about 0.5 mg/mL, at least about 0.6 mg/mL, atleast about 0.7 mg/mL, at least about 0.8 mg/mL, at least about 0.9mg/mL, at least about 1 mg/mL, at least about 2 mg/mL, at least about 3mg/mL, at least about 4 mg/mL, at least about 5 mg/mL, at least about 10mg/mL, at least about 15 mg/mL, at least about 20 mg/mL, at least about25 mg/mL, at least about 30 mg/mL, at least about 35 mg/mL, at leastabout 40 mg/mL, at least about 45 mg/mL, at least about 50 mg/mL, atleast about 55 mg/mL, at least about 60 mg/mL, at least about 65 mg/mL,at least about 70 mg/mL, at least about 75 mg/mL, at least about 80mg/mL, at least about 85 mg/mL, at least about 90 mg/mL, at least about95 mg/mL, at least about 100 mg/mL, at least about 125 mg/mL, at leastabout 150 mg/mL, at least about 175 mg/mL, at least about 200 mg/mL, atleast about 250 mg/mL, at least about 300 mg/mL, at least about 350mg/mL, at least about 400 mg/mL, at least about 450 mg/mL, at leastabout 500 mg/mL, at least about 550 mg/mL, at least about 600 mg/mL, atleast about 650 mg/mL, at least about 700 mg/mL, at least about 750mg/mL, at least about 800 mg/mL, at least about 850 mg/mL, at leastabout 900 mg/mL, at least about 950 mg/mL, at least about 1000 mg/mL, atleast about 1050 mg/mL, at least about 1100 mg/mL, at least about 1150mg/mL, at least about 1200 mg/mL, at least about 1250 mg/mL, at leastabout 1300 mg/mL, at least about 1350 mg/mL, at least about 1400 mg/mL,at least about 1450 mg/mL, at least about 1500 mg/mL, at least about1550 mg/mL, at least about 1600 mg/mL, at least about 1650 mg/mL, atleast about 1700 mg/mL, at least about 1750 mg/mL, at least about 1800mg/mL, at least about 1850 mg/mL, at least about 1900 mg/mL, at leastabout 1950 mg/mL, or at least about 2000 mg/mL.

In some embodiments, a formulation of the disclosure comprises acompound of the disclosure at a concentration of at most about 0.002mg/mL, at most about 0.003 mg/mL, at most about 0.004 mg/mL, at mostabout 0.005 mg/mL, at most about 0.006 mg/mL, at most about 0.007 mg/mL,at most about 0.008 mg/mL, at most about 0.009 mg/mL, at most about 0.01mg/mL, at most about 0.02 mg/mL, at most about 0.03 mg/mL, at most about0.04 mg/mL, at most about 0.05 mg/mL, at most about 0.06 mg/mL, at mostabout 0.07 mg/mL, at most about 0.08 mg/mL, at most about 0.09 mg/mL, atmost about 0.1 mg/mL, at most about 0.2 mg/mL, at most about 0.3 mg/mL,at most about 0.4 mg/mL, at most about 0.5 mg/mL, at most about 0.6mg/mL, at most about 0.7 mg/mL, at most about 0.8 mg/mL, at most about0.9 mg/mL, at most about 1 mg/mL, at most about 2 mg/mL, at most about 3mg/mL, at most about 4 mg/mL, at most about 5 mg/mL, at most about 10mg/mL, at most about 15 mg/mL, at most about 20 mg/mL, at most about 25mg/mL, at most about 30 mg/mL, at most about 35 mg/mL, at most about 40mg/mL, at most about 45 mg/mL, at most about 50 mg/mL, at most about 55mg/mL, at most about 60 mg/mL, at most about 65 mg/mL, at most about 70mg/mL, at most about 75 mg/mL, at most about 80 mg/mL, at most about 85mg/mL, at most about 90 mg/mL, at most about 95 mg/mL, at most about 100mg/mL, at most about 125 mg/mL, at most about 150 mg/mL, at most about175 mg/mL, at most about 200 mg/mL, at most about 250 mg/mL, at mostabout 300 mg/mL, at most about 350 mg/mL, at most about 400 mg/mL, atmost about 450 mg/mL, at most about 500 mg/mL, at most about 550 mg/mL,at most about 600 mg/mL, at most about 650 mg/mL, at most about 700mg/mL, at most about 750 mg/mL, at most about 800 mg/mL, at most about850 mg/mL, at most about 900 mg/mL, at most about 950 mg/mL, at mostabout 1000 mg/mL, at most about 1050 mg/mL, at most about 1100 mg/mL, atmost about 1150 mg/mL, at most about 1200 mg/mL, at most about 1250mg/mL, at most about 1300 mg/mL, at most about 1350 mg/mL, at most about1400 mg/mL, at most about 1450 mg/mL, at most about 1500 mg/mL, at mostabout 1550 mg/mL, at most about 1600 mg/mL, at most about 1650 mg/mL, atmost about 1700 mg/mL, at most about 1750 mg/mL, at most about 1800mg/mL, at most about 1850 mg/mL, at most about 1900 mg/mL, at most about1950 mg/mL, or at most about 2000 mg/mL.

In some embodiments, a formulation of the disclosure comprises acompound of the disclosure at a concentration of about 1 mg/mL to about2000 mg/mL; from about 5 mg/mL to about 1000 mg/mL, from about 10 mg/mLto about 25 mg/mL, from about 50 mg/mL to about 250 mg/mL, from about100 mg/mL to about 200 mg/mL, from about 1 mg/mL to about 50 mg/mL, fromabout 50 mg/mL to about 100 mg/mL, from about 100 mg/mL to about 150mg/mL, from about 150 mg/mL to about 200 mg/mL, from about 200 mg/mL toabout 250 mg/mL, from about 250 mg/mL to about 300 mg/mL, from about 300mg/mL to about 350 mg/mL, from about 350 mg/mL to about 400 mg/mL, fromabout 400 mg/mL to about 450 mg/mL, from about 450 mg/mL to about 500mg/mL, from about 500 mg/mL to about 550 mg/mL, from about 550 mg/mL toabout 600 mg/mL, from about 600 mg/mL to about 650 mg/mL, from about 650mg/mL to about 700 mg/mL, from about 700 mg/mL to about 750 mg/mL, fromabout 750 mg/mL to about 800 mg/mL, from about 800 mg/mL to about 850mg/mL, from about 850 mg/mL to about 900 mg/mL, from about 900 mg/mL toabout 950 mg/mL, from about 950 mg/mL to about 1000 mg/mL, about 1 μg/mLto about 2000 μg/mL; from about 5 μg/mL to about 1000 μg/mL, from about10 μg/mL to about 25 μg/mL, from about 50 μg/mL to about 250 μg/mL, fromabout 100 μg/mL to about 200 μg/mL, from about 1 μg/mL to about 50μg/mL, from about 50 μg/mL to about 100 μg/mL, from about 100 μg/mL toabout 150 μg/mL, from about 150 μg/mL to about 200 μg/mL, from about 200μg/mL to about 250 μg/mL, from about 250 μg/mL to about 300 μg/mL, fromabout 300 μg/mL to about 350 μg/mL, from about 350 μg/mL to about 400μg/mL, from about 400 μg/mL to about 450 μg/mL, from about 450 μg/mL toabout 500 μg/mL, from about 500 μg/mL to about 550 μg/mL, from about 550μg/mL to about 600 μg/mL, from about 600 μg/mL to about 650 μg/mL, fromabout 650 μg/mL to about 700 μg/mL, from about 700 μg/mL to about 750μg/mL, from about 750 μg/mL to about 800 μg/mL, from about 800 μg/mL toabout 850 μg/mL, from about 850 μg/mL to about 900 μg/mL, from about 900μg/mL to about 950 μg/mL, or from about 950 μg/mL to about 1000 μg/mL.

Therapeutic Effects Tissue Distribution and Pharmacokinetics

Compounds disclosed herein can have favorable properties foradministration to subjects or patients, for example, favorablepharmacokinetic or tissue distribution parameters.

In some embodiments, if a study or assay (e.g., tissue distributionstudy) is conducted, wherein the study or assay comprises administering(e.g., intracerebroventricular administration) a compound of thedisclosure (e.g., Compound 1) to a study patient or subject at a doseamount of about 0.1 mg/kg to about 1.5 mg/kg, then the compoundaccumulates in the study patient's brain for at most about 1 month afterdosing. In some embodiments, the compound is not observed or is rarelyobserved at a detectable level during the month in the study patient'splasma, intestine, liver, lung, kidney, and/or muscle.

In some embodiments, the compound can be subjected to an assay or astudy (e.g., a tissue distribution study) and in the study, the compoundcan be observed to accumulate in the brain of a study patient (e.g.,mice) for a time period (e.g., most about a month) after theadministering. In some embodiments, the compound is not observed at adetectable level during the time period in the plasma, intestine, liver,lung, kidney, and/or muscle of the study patient. In some embodiments,the assay can comprise administering (e.g., intracerebroventricularadministration) a dose amount (e.g., about 0.1 mg/kg to about 1.5 mg/kg)of the compound to a study patient (e.g., mice). Blood samples can becollected (e.g., from cava veins) of the study patient at a time point(e.g., between about 1 hour and 28 days) post administration. Aftercollecting the blood samples, the study patients can be euthanized at atime point between (e.g., about 1 hour and 28 days) post administration.After the euthanasia, various tissues (e.g., brain, intestine, liver,lung, kidney, and/or muscle tissues) can be collected from the studypatients.

Various analytical techniques can be used to determine concentrations ofthe compound in tissues and/or other samples collected from studypatients (for example, blood, plasma, urine, feces, etc). Non-limitingexamples of techniques that can be used to determine concentrations ofthe compound include mass spectrometry, for example, liquidchromatography mass spectrometry (LC-MS), gas chromatography massspectrometry (GC-MS), tandem MS (MS/MS, e.g, LC-MS/MS or GC-MS/MS),Matrix-assisted laser desorption ionization-time of flight massspectrometry (MALDI-TOF MS), triple quadrupole mass spectrometry (TQMS),Quadrupole Trap MS, hybrid linear trap orbitrap MS, quadrupole-Orbitrapmass spectrometry, High performance or ultra-high performance liquidchromatography (HPLC or UHPLC, e.g., with MS or ultraviolet detection),time of flight (TOF) MS, Selected reaction monitoring (SRM), Multiplereaction monitoring (MRM) nuclear magnetic resonance (NMR, for example,continuous-wave (cw), pulsed or Fourier-Transform, 1H, 13C, 19F, 31P, orother nuclei), variations thereof, or combinations thereof.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient (e.g., mouse) or subject at a doseamount of about 0.01 mg/kg to about 5 mg/kg (for example, about 0.1 toabout 2 mg/kg, about 0.2 to about 1.5 mg/kg, about 0.3 to about 1.2mg/kg, about 0.3 to about 1 mg/kg, about 0.3 to about 0.6 mg/kg, orabout 0.6 to about 1 mg/kg), then the compound accumulates in the studypatient's or subject's brain for a time period of at most about 1 month(for example, at most about 3 days, at most about 7 days, at most about14 days, at most about 21 days, at most about 27 days, or at most about28 days) after dosing. In some embodiments, the compound is not observedor is rarely observed during this time period at a detectable level inthe study patient's or subject's plasma, intestine, liver, lung, kidney,and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.01 mg/kg to about 5 mg/kg (for example, about 0.1 to about 2mg/kg, about 0.2 to about 1.5 mg/kg, about 0.3 to about 1.2 mg/kg, about0.3 to about 1 mg/kg, about 0.3 to about 0.6 mg/kg, or about 0.6 toabout 1 mg/kg), then the compound accumulates in the study patient's orsubject's brain for a time period of at least about 1 day (for example,at least about 1 day, at least about 3 days, at least about 7 days, atleast about 14 days, at least about 21 days, at least about 27 days, atleast about 28 days, at least about 1 month, at least about 2 months, orat least about 3 months) after dosing. In some embodiments, the compoundis not observed or is rarely observed during this time period at adetectable level in the study patient's or subject's plasma, intestine,liver, lung, kidney, and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.01 mg/kg to about 5 mg/kg (for example, about 0.1 to about 2mg/kg, about 0.2 to about 1.5 mg/kg, about 0.3 to about 1.2 mg/kg, about0.3 to about 1 mg/kg, about 0.3 to about 0.6 mg/kg, or about 0.6 toabout 1 mg/kg), then the compound accumulates in the study patient's orsubject's spleen for a time period of at most about 1 month (forexample, at most about 3 days, at most about 7 days, at most about 14days, at most about 21 days, at most about 27 days, or at most about 28days) after dosing. In some embodiments, the compound is not observed oris rarely observed at a detectable level during this time period in thestudy patient's or subject's plasma, intestine, liver, lung, kidney,and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.01 mg/kg to about 5 mg/kg (for example, about 0.1 to about 2mg/kg, about 0.2 to about 1.5 mg/kg, about 0.3 to about 1.2 mg/kg, about0.3 to about 1 mg/kg, about 0.3 to about 0.6 mg/kg, or about 0.6 toabout 1 mg/kg), then the compound accumulates in the study patient's orsubject's spleen for a time period of at least about 1 day (for example,at least about 1 day, at least about 3 days, at least about 7 days, atleast about 14 days, at least about 21 days, at least about 27 days, atleast about 28 days, at least about 1 month, at least about 2 months, orat least about 3 months) after dosing. In some embodiments, the compoundis not observed or is rarely observed at a detectable level during thistime period in the study patient's or subject's plasma, intestine,liver, lung, kidney, and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.01 mg/kg to about 5 mg/kg (for example, about 0.1 to about 2mg/kg, about 0.2 to about 1.5 mg/kg, about 0.3 to about 1.2 mg/kg, about0.3 to about 1 mg/kg, about 0.3 to about 0.6 mg/kg, or about 0.6 toabout 1 mg/kg), then the compound accumulates in the study patient's orsubject's heart for a timer period of at most about 1 month (forexample, at most about 3 days, at most about 7 days, at most about 14days, at most about 21 days, at most about 27 days, or at most about 28days) after dosing. In some embodiments, the compound is not observed oris rarely observed at a detectable level during this time period in thestudy patient's or subject's plasma, intestine, liver, lung, kidney,and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.01 mg/kg to about 5 mg/kg (for example, about 0.1 to about 2mg/kg, about 0.2 to about 1.5 mg/kg, about 0.3 to about 1.2 mg/kg, about0.3 to about 1 mg/kg, about 0.3 to about 0.6 mg/kg, or about 0.6 toabout 1 mg/kg), then the compound accumulates in the study patient's orsubject's heart for a time period of at least about 1 day (for example,at least about 1 day, at least about 3 days, at least about 7 days, atleast about 14 days, at least about 21 days, at least about 27 days, atleast about 28 days, at least about 1 month, at least about 2 months, orat least about 3 months) after dosing. In some embodiments, the compoundis not observed or is rarely observed at a detectable level during thisperiod in the study patient's or subject's plasma, intestine, liver,lung, kidney, and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.001 mg/kg to about 0.5 mg/kg (for example, about 0.008 to about0.163 mg/kg, about 0.016 to about 0.125 mg/kg, about 0.025 to about 0.1mg/kg, about 0.025 to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg,or about 0.05 to about 0.08 mg/kg), then the compound accumulates in thestudy patient's or subject's brain for a time period of at most about 1month (for example, at most about 3 days, at most about 7 days, at mostabout 14 days, at most about 21 days, at most about 27 days, or at mostabout 28 days) after dosing. In some embodiments, the compound is notobserved or is rarely observed at a detectable level during this timeperiod in the study patient's or subject's plasma, intestine, liver,lung, kidney, and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.001 mg/kg to about 0.5 mg/kg (for example, about 0.008 to about0.163 mg/kg, about 0.016 to about 0.125 mg/kg, about 0.025 to about 0.1mg/kg, about 0.025 to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg,or about 0.05 to about 0.08 mg/kg), then the compound accumulates in thestudy patient's or subject's brain at a time period for at least about 1day (for example, at least about 1 day, at least about 3 days, at leastabout 7 days, at least about 14 days, at least about 21 days, at leastabout 27 days, at least about 28 days, at least about 1 month, at leastabout 2 months, or at least about 3 months) after dosing. In someembodiments, the compound is not observed or is rarely observed at adetectable level during this time period in the study patient's orsubject's plasma, intestine, liver, lung, kidney, and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.001 mg/kg to about 0.5 mg/kg (for example, about 0.008 to about0.163 mg/kg, about 0.016 to about 0.125 mg/kg, about 0.025 to about 0.1mg/kg, about 0.025 to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg,or about 0.05 to about 0.08 mg/kg), then the compound accumulates in thestudy patient's or subject's spleen for a time period of at most about 1month (for example, at most about 3 days, at most about 7 days, at mostabout 14 days, at most about 21 days, at most about 27 days, or at mostabout 28 days) after dosing. In some embodiments, the compound is notobserved or is rarely observed during this time period at a detectablelevel in the study patient's or subject's plasma, intestine, liver,lung, kidney, and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.001 mg/kg to about 0.5 mg/kg (for example, about 0.008 to about0.163 mg/kg, about 0.016 to about 0.125 mg/kg, about 0.025 to about 0.1mg/kg, about 0.025 to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg,or about 0.05 to about 0.08 mg/kg), then the compound accumulates in thestudy patient's or subject's spleen for a time period of at least about1 day (for example, at least about 1 day, at least about 3 days, atleast about 7 days, at least about 14 days, at least about 21 days, atleast about 27 days, at least about 28 days, at least about 1 month, atleast about 2 months, or at least about 3 months) after dosing. In someembodiments, the compound is not observed or is rarely observed at adetectable level during this time period in the study patient's orsubject's plasma, intestine, liver, lung, kidney, and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.001 mg/kg to about 0.5 mg/kg (for example, about 0.008 to about0.163 mg/kg, about 0.016 to about 0.125 mg/kg, about 0.025 to about 0.1mg/kg, about 0.025 to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg,or about 0.05 to about 0.08 mg/kg), then the compound accumulates in thestudy patient's or subject's heart for a time period of at most about 1month (for example, at most about 3 days, at most about 7 days, at mostabout 14 days, at most about 21 days, at most about 27 days, or at mostabout 28 days) after dosing. In some embodiments, the compound is notobserved or is rarely observed at a detectable level during this timeperiod in the study patient's or subject's plasma, intestine, liver,lung, kidney, and/or muscle.

In some embodiments, if an assay or study is conducted, wherein thestudy or assay comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure(e.g., Compound 1) to a study patient or subject at a dose amount ofabout 0.001 mg/kg to about 0.5 mg/kg (for example, about 0.008 to about0.163 mg/kg, about 0.016 to about 0.125 mg/kg, about 0.025 to about 0.1mg/kg, about 0.025 to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg,or about 0.05 to about 0.08 mg/kg), then the compound accumulates in thestudy patient's or subject's heart for a time period of at least about 1day (for example, at least about 1 day, at least about 3 days, at leastabout 7 days, at least about 14 days, at least about 21 days, at leastabout 27 days, at least about 28 days, at least about 1 month, at leastabout 2 months, or at least about 3 months) after dosing. In someembodiments, the compound is not observed or is rarely observed at adetectable level during this time period in the study patient's orsubject's plasma, intestine, liver, lung, kidney, and/or muscle.

In some embodiments, a dose can be modulated to achieve a desiredpharmacokinetic or pharmacodynamics profile, such as a desired oreffective blood profile, as described herein.

Pharmacokinetic and pharmacodynamic data can be obtained by variousexperimental techniques. Appropriate pharmacokinetic and pharmacodynamicprofile components describing a particular composition can vary due tovariations in drug metabolism in human subjects. Pharmacokinetic andpharmacodynamic profiles can be based on the determination of the meanparameters of a group of subjects. The group of subjects includes anyreasonable number of subjects suitable for determining a representativemean, e.g., 5 subjects, 10 subjects, 15 subjects, 20 subjects, 25subjects, 30 subjects, 35 subjects, or more. The mean is determined, forexample, by calculating the average of all subject's measurements foreach parameter measured. A dose can be modulated to achieve a desiredpharmacokinetic or pharmacodynamics profile, such as a desired oreffective blood profile, as described herein.

In some embodiments, the subject is a vertebrate. In some embodiments,the subject is a mammal. In some embodiments, the subject is a human. Insome embodiments, the subject is a primate, ape, monkey, sheep, equine,bovine, porcine, minipig, canine, feline, goat, camelid, rodent, rabbit,mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, guinea pig,C57BL6J mouse, Beagle dog, Gottingen minipig, or Cynomolgus monkey. Insome embodiments, a subject is a non-human subject. In some embodiments,a subject is a veterinary subject.

In some embodiments, the patient is a vertebrate. In some embodiments,the patient is a mammal. In some embodiments, the patient is a human. Insome embodiments, the patient is a primate, ape, monkey, sheep, equine,bovine, porcine, minipig, canine, feline, goat, camelid, rodent, rabbit,mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, guinea pig,C57BL6J mouse, Beagle dog, Gottingen minipig, or Cynomolgus monkey. Insome embodiments, a patient is a non-human patient. In some embodiments,a patient is a veterinary patient.

In some embodiments, a patient and a subject are the same species. Insome embodiments, a subject and a patient are human.

In some embodiments, a patient and a subject are different species. Insome embodiments, a subject is human and a patient is a non-human, forexample, a non-human vertebrate, non-human mammal, non-human primate,ape, monkey, sheep, equine, bovine, porcine, minipig, canine, feline,goat, camelid, rodent, rabbit, mouse, rat, hamster, gerbil, hamster,chinchilla, fancy rat, or guinea pig. In some embodiments, a patient ishuman and a subject is a non-human, for example, a non-human vertebrate,non-human mammal, non-human primate, ape, monkey, sheep, equine, bovine,porcine, minipig, canine, feline, goat, camelid, rodent, rabbit, mouse,rat, hamster, gerbil, hamster, chinchilla, fancy rat, or guinea pig.

A pharmacokinetic parameter can be any parameter suitable for describinga compound. Non-limiting examples of pharmacodynamic and pharmacokineticparameters that can be calculated for a compound that is administeredwith the methods of the invention include:

a) the amount of drug administered, which can be represented as a doseD;b) the dosing interval, which can be represented as τ;c) the apparent volume in which a drug is distributed, which can berepresented as a volume of distribution V_(d), where V_(d)=D/C₀;d) the amount of drug in a given volume of plasma, which can berepresented as concentration C₀ or C_(ss), where C₀ or C_(ss)=D/Vd andcan be represented as a mean plasma concentration over a plurality ofsamples;e) the half-life of a drug t_(1/2), where t_(1/2)=ln(2)/k_(e);f) the rate at which a drug is removed from the body k_(e), wherek_(e)=ln(2)/t_(1/2)=CL/V_(d); g) the rate of infusion required tobalance the equation K_(in), where K_(in)=C_(ss)·CL;h) the integral of the concentration-time curve after administration ofa single dose, which can be represented as AUC_(0-∞), wherein ∫₀ ^(∞)Cdt, or in steady-state, which can be represented as AUCτ, _(ss), wherein∫_(t) ^(t+π)C dt; i) the volume of plasma cleared of the drug per unittime, which can be represented as CL (clearance), whereinCL=V_(d)·k_(e)=D/AUC;j) the systemically available fraction of a drug, which can berepresented as f, where

${f = \frac{{AUCpo}.{Div}}{{AUCiv}.{Dpo}}};$

k) the peak plasma concentration of a drug after administration,C_(max);l) the time taken by a drug to reach C_(max), t_(max);m) the lowest concentration that a drug reaches before the next dose isadministered C_(min); andn) the peak trough fluctuation within one dosing interval at steadystate, which can be represented as

${\%\mspace{20mu}{PTF}} = {{{100 \cdot \frac{\left( {{C\;\max,{ss}} - {C\;\min,{ss}}} \right)}{{Cav},{ss}}}\mspace{14mu}{where}\mspace{14mu} C_{{av},{ss}}} = \frac{{AUC}\;\tau,{ss}}{\tau}}$

In some embodiments, if the compound is subjected to a study or an assay(e.g., a pharmacokinetic study), then a mean maximum brain concentration(e.g., 3000 ng/mL to about 22000 ng/mL) can be observed in a studypatient (e.g., mice) at a time to maximum brain concentration (e.g.,about 1 hour to about 50 hours post administration). In someembodiments, the assay can comprise administering by administering(e.g., intracerebroventricular administration) a dose amount of about0.1 mg/kg to about 2 mg/kg of the compound to the study patient (e.g.,mice). The mice can be euthanized at a time point (e.g., between about 1hour and 28 days) post administration. Various tissues (e.g., braintissues) can be collected from the study patients after the euthanizing.Concentrations of the tissues in the study patients can be determinedusing various techniques, disclosed herein, such as liquidchromatography-tandem mass spectrometry.

Brain Pharmacokinetics

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(max) of the compound in brain is at least 0.5, at least 1, at least2, at least 4, at least 8, at least 12, at least 16, at least 20, atleast 24, at least 28, at least 32, at least 36, at least 40, at least44, or at least 48 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(max) of the compound in brain is at most 6, at most 12, at most 16,at most 20, at most 24, at most 28, at most 32, at most 36, at most 40,at most 44, at most 48, at most 72, at most 96, or at most 120 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(max) of the compound in brain is about 0.5 to about 120, about 1 toabout 24, about 1 to about 36, about 1 to about 48, about 2 to about 36,about 6 to about 24, about 1 to about 6, about 6 to about 12, about 12to about 24, about 12 to about 18, about 18 to about 48, about 18 toabout 36, or about 24 to about 48 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanC_(max) of the compound in brain is at least about 1, at least about1000, at least about 2000, at least about 3000, at least about 4000, atleast about 5000, at least about 6000, at least about 7000, at leastabout 8000, at least about 9000, at least about 10000, at least about12000, at least about 14000, at least about 16000, at least about 18000,at least about 20000, at least about 22000, or at least about 25000ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanCmax of the compound in brain is at most about 3000, at most about 4000,at most about 5000, at most about 6000, at most about 7000, at mostabout 8000, at most about 9000, at most about 10000, at most about12000, at most about 14000, at most about 16000, at most about 18000, atmost about 20000, at most about 22000, at most about 25000, at mostabout 30000 or at most about 50000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanCmax of the compound in brain is about 1000 to about 50000, about 2000to about 25000, about 3000 to about 22000, about 2000 to about 5000,about 5000 to about 15000, or about 10000 to about 25000 ng/mL.

In some embodiments, at a dose amount of 0.3 mg/kg, the mean maximumbrain concentration is from about 3000 ng/mL to about 4000 ng/mL. Insome embodiments, at a dose amount of 0.6 mg/kg, the mean maximum brainconcentration is from about 6000 ng/mL to about 12000 ng/mL. In someembodiments, at a dose amount of 1 mg/kg, the mean maximum brainconcentration is from about 15000 ng/mL to about 22000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanAUC_(last) of the compound for brain is at least about 0.5×10{circumflexover ( )}6, at least about 1×10{circumflex over ( )}6, at least about1.5×10{circumflex over ( )}6, at least about 2×10{circumflex over ( )}6,at least about 2.5×10{circumflex over ( )}6, at least about3×10{circumflex over ( )}6, at least about 4×10{circumflex over ( )}6,at least about 5×10{circumflex over ( )}6, at least about6×10{circumflex over ( )}6, at least about 7×10{circumflex over ( )}6,at least about 8×10{circumflex over ( )}6, or at least about10×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanAUClast of the compound for brain is at most about 1.5×10{circumflexover ( )}6, at most about 2×10{circumflex over ( )}6, at most about2.5×10{circumflex over ( )}6, at most about 3×10{circumflex over ( )}6,at most about 4×10{circumflex over ( )}6, at most about 5×10{circumflexover ( )}6, at most about 6×10{circumflex over ( )}6, at most about7×10{circumflex over ( )}6, at most about 8×10{circumflex over ( )}6, atmost about 10×10{circumflex over ( )}6, at most about 15×10{circumflexover ( )}6, or at most about 20×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanAUClast of the compound for brain is about 0.5×10{circumflex over ( )}6to about 20×10{circumflex over ( )}6, about 1×10{circumflex over ( )}6to about 15×10{circumflex over ( )}6, about 1×10{circumflex over ( )}6to about 10×10{circumflex over ( )}6, about 1.5×10{circumflex over ( )}6to about 8×10{circumflex over ( )}6, about 1×10{circumflex over ( )}6 toabout 5×10{circumflex over ( )}6, about 5×10{circumflex over ( )}6 toabout 10×10{circumflex over ( )}6, about 1×10{circumflex over ( )}6 toabout 3×10{circumflex over ( )}6, or about 2×10{circumflex over ( )}6 toabout 5×10{circumflex over ( )}6 ng·h/mL. In some embodiments, theAUClast of the study patient or subject (e.g., mice) is observed to beabout 1400000 h*ng/mL to about 7500000 h*ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanTlast for brain is at least about 1, at least about 2, at least about 3,at least about 4, at least about 5, at least about 6, at least about 7,at least about 10, at least about 12, at least about 14, at least about21, at least about 28, at least about 35, at least about 42, at leastabout 49, or at least about 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanTlast for brain is at most about 1, at most about 2, at most about 3, atmost about 4, at most about 5, at most about 6, at most about 7, at mostabout 10, at most about 12, at most about 14, at most about 21, at mostabout 28, at most about 35, at most about 42, at most about 49, at mostabout 100 days, at most about 200, or at most about 300 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanTlast for brain is about 1 to about 300, about 2 to about 100, about 5to about 50, about 10 to about 30, about 1 to about 20, about 20 toabout 40, about 40 to about 60, about 60 to about 80, or about 80 toabout 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(1/2) for brain is at least 6, at least 24, at least 48, at least 100,at least 200, at least 500, at least 1000, at least 1500, at least 2000,at least 2500, at least 3000, or at least 5000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the mean T½for brain is at most 24, at most 48, at most 100, at most 200, at most500, at most 1000, at most 1500, at most 2000, at most 2500, at most3000, at most 5000, at most 10000, or at most 20000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the mean T½for brain is about 24 to about 20000, about 100 to about 15000, about500 to about 10000, about 500 to about 5000, about 100 to about 1000,about 1000 to about 2000, or about 3000 to about 5000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(max) of the compound in brain is atleast 0.5, at least 1, at least 2, at least 4, at least 8, at least 12,at least 16, at least 20, at least 24, at least 28, at least 32, atleast 36, at least 40, at least 44, or at least 48 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(max) of the compound in brain is atmost 6, at most 12, at most 16, at most 20, at most 24, at most 28, atmost 32, at most 36, at most 40, at most 44, at most 48, at most 72, atmost 96, or at most 120 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(max) of the compound in brain isabout 0.5 to about 120, about 1 to about 24, about 1 to about 36, about1 to about 48, about 2 to about 36, about 6 to about 24, about 1 toabout 6, about 6 to about 12, about 12 to about 24, about 12 to about18, about 18 to about 48, about 18 to about 36, or about 24 to about 48hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean C_(max) of the compound in brain is atleast about 1, at least about 1000, at least about 2000, at least about3000, at least about 4000, at least about 5000, at least about 6000, atleast about 7000, at least about 8000, at least about 9000, at leastabout 10000, at least about 12000, at least about 14000, at least about16000, at least about 18000, at least about 20000, at least about 22000,or at least about 25000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Cmax of the compound in brain is atmost about 3000, at most about 4000, at most about 5000, at most about6000, at most about 7000, at most about 8000, at most about 9000, atmost about 10000, at most about 12000, at most about 14000, at mostabout 16000, at most about 18000, at most about 20000, at most about22000, at most about 25000, at most about 30000 or at most about 50000ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Cmax of the compound in brain is about1000 to about 50000, about 2000 to about 25000, about 3000 to about22000, about 2000 to about 5000, about 5000 to about 15000, or about10000 to about 25000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean AUC_(last) of the compound for brain isat least about 0.5×10{circumflex over ( )}6, at least about1×10{circumflex over ( )}6, at least about 1.5×10{circumflex over ( )}6,at least about 2×10{circumflex over ( )}6, at least about2.5×10{circumflex over ( )}6, at least about 3×10{circumflex over ( )}6,at least about 4×10{circumflex over ( )}6, at least about5×10{circumflex over ( )}6, at least about 6×10{circumflex over ( )}6,at least about 7×10{circumflex over ( )}6, at least about8×10{circumflex over ( )}6, or at least about 10×10{circumflex over( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean AUClast of the compound for brain is atmost about 1.5×10{circumflex over ( )}6, at most about 2×10{circumflexover ( )}6, at most about 2.5×10{circumflex over ( )}6, at most about3×10{circumflex over ( )}6, at most about 4×10{circumflex over ( )}6, atmost about 5×10{circumflex over ( )}6, at most about 6×10{circumflexover ( )}6, at most about 7×10{circumflex over ( )}6, at most about8×10{circumflex over ( )}6, at most about 10×10{circumflex over ( )}6,at most about 15×10{circumflex over ( )}6, or at most about20×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean AUClast of the compound for brain isabout 0.5×10{circumflex over ( )}6 to about 20×10{circumflex over ( )}6,about 1×10{circumflex over ( )}6 to about 15×10{circumflex over ( )}6,about 1×10{circumflex over ( )}6 to about 10×10{circumflex over ( )}6,about 1.5×10{circumflex over ( )}6 to about 8×10{circumflex over ( )}6,about 1×10{circumflex over ( )}6 to about 5×10{circumflex over ( )}6,about 5×10{circumflex over ( )}6 to about 10×10{circumflex over ( )}6,about 1×10{circumflex over ( )}6 to about 3×10{circumflex over ( )}6, orabout 2×10{circumflex over ( )}6 to about 5×10{circumflex over ( )}6ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Tlast for brain is at least about 1, atleast about 2, at least about 3, at least about 4, at least about 5, atleast about 6, at least about 7, at least about 10, at least about 12,at least about 14, at least about 21, at least about 28, at least about35, at least about 42, at least about 49, or at least about 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Tlast for brain is at most about 1, atmost about 2, at most about 3, at most about 4, at most about 5, at mostabout 6, at most about 7, at most about 10, at most about 12, at mostabout 14, at most about 21, at most about 28, at most about 35, at mostabout 42, at most about 49, at most about 100 days, at most about 200,or at most about 300 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Tlast for brain is about 1 to about300, about 2 to about 100, about 5 to about 50, about 10 to about 30,about 1 to about 20, about 20 to about 40, about 40 to about 60, about60 to about 80, or about 80 to about 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T½ for brain is at least 6, at least24, at least 48, at least 100, at least 200, at least 500, at least1000, at least 1500, at least 2000, at least 2500, at least 3000, or atleast 5000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T½ for brain is at most 24, at most 48,at most 100, at most 200, at most 500, at most 1000, at most 1500, atmost 2000, at most 2500, at most 3000, at most 5000, at most 10000, orat most 20000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T½ for brain is about 24 to about20000, about 100 to about 15000, about 500 to about 10000, about 500 toabout 5000, about 100 to about 1000, about 1000 to about 2000, or about3000 to about 5000 hours.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 1.5 hour at 0.3mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 48 hour at 0.3 mg/kg,after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 4 hour at 0.6 mg/kg,after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 1.5 hour at 0.6mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 24 hour at 1 mg/kg,after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 8 hour at 1 mg/kg,after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 3110 ng/mL at 0.3mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 3930 ng/mL at 0.3mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 6490 ng/mL at 0.6mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 11400 ng/mL at 0.6mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 15500 ng/mL at 1mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 21500 ng/mL at 1mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 1440000ng·h/mL at 0.3 mg/kg, after administration of a single dose to a malesubject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 1800000ng·h/mL at 0.3 mg/kg, after administration of a single dose to a femalesubject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 2470000ng·h/mL at 0.6 mg/kg, after administration of a single dose to a malesubject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 2360000ng·h/mL at 0.6 mg/kg, after administration of a single dose to a femalesubject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 5530000ng·h/mL at 1 mg/kg, after administration of a single dose to a malesubject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 7150000ng·h/mL at 1 mg/kg, after administration of a single dose to a femalesubject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(last) ofthe compound is within 70% to 130% of a T_(last) of 648 hours, afteradministration of a single dose to a subject.

Spleen Pharmacokinetics

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(max) of the compound in spleen is at least 0.5, at least 1, at least2, at least 4, at least 8, at least 12, at least 16, at least 20, atleast 24, at least 28, at least 32, at least 36, at least 40, at least44, or at least 48 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(max) of the compound in spleen is at most 6, at most 12, at most 16,at most 20, at most 24, at most 28, at most 32, at most 36, at most 40,at most 44, at most 48, at most 72, at most 96, or at most 120 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(max) of the compound in spleen is about 0.5 to about 120, about 1 toabout 24, about 1 to about 36, about 1 to about 48, about 2 to about 36,about 6 to about 24, about 1 to about 6, about 6 to about 12, about 12to about 24, about 12 to about 18, about 18 to about 48, about 18 toabout 36, or about 24 to about 48 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanC_(max) of the compound in spleen is at least about 1, at least about50, at least about 100, at least about 250, at least about 500, at leastabout 750, at least about 1250, at least about 1500, at least about2000, at least about 2500, at least about 3000, at least about 5000, orat least about 10000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanCmax of the compound in spleen is at most about 1000, at most about1500, at most about 2000, at most about 2500, at most about 3000, atmost about 4000, at most about 5000, or at most about 10000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanCmax of the compound in spleen is about 100 to about 50000, about 200 toabout 5000, about 300 to about 4000, about 500 to about 3000, about 500to about 1000, about 1000 to about 2000, about 1000 to about 3000, orabout 2000 to about 4000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanAUC_(last) of the compound for spleen is at least about0.01×10{circumflex over ( )}6, at least about 0.05×10{circumflex over( )}6, at least about 0.08×10{circumflex over ( )}6, at least about0.1×10{circumflex over ( )}6, at least about 0.2×10{circumflex over( )}6, at least about 0.3×10{circumflex over ( )}6, at least about0.4×10{circumflex over ( )}6, at least about 0.5×10{circumflex over( )}6, at least about 0.75×10{circumflex over ( )}6, at least about1×10{circumflex over ( )}6, at least about 3×10{circumflex over ( )}6,or at least about 5×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanAUClast of the compound for spleen is at most about 0.08×10{circumflexover ( )}6, at most about 0.1×10{circumflex over ( )}6, at most about0.2×10{circumflex over ( )}6, at most about 0.3×10{circumflex over( )}6, at most about 0.4×10{circumflex over ( )}6, at most about0.5×10{circumflex over ( )}6, at most about 0.75×10{circumflex over( )}6, at most about 1×10{circumflex over ( )}6, at most about1.5×10{circumflex over ( )}6, at most about 2×10{circumflex over ( )}6,or at most about 5×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanAUClast of the compound for spleen is about 0.01×10{circumflex over( )}6 to about 5×10{circumflex over ( )}6, about 0.05×10{circumflex over( )}6 to about 3×10{circumflex over ( )}6, about 0.075×10{circumflexover ( )}6 to about 2×10{circumflex over ( )}6, about 0.09×10{circumflexover ( )}6 to about 1.1×10{circumflex over ( )}6, about0.1×10{circumflex over ( )}6 to about 0.8×10{circumflex over ( )}6,about 0.1×10{circumflex over ( )}6 to about 0.5×10{circumflex over( )}6, about 0.1×10{circumflex over ( )}6 to about 0.3×10{circumflexover ( )}6, about 0.3×10{circumflex over ( )}6 to about 1×10{circumflexover ( )}6, about 0.5×10{circumflex over ( )}6 to about 1×10{circumflexover ( )}6, about 0.1×10{circumflex over ( )}6 to about0.2×10{circumflex over ( )}6, about 0.2×10{circumflex over ( )}6 toabout 0.3×10{circumflex over ( )}6, or about 0.3×10{circumflex over( )}6 to about 0.5×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanTlast for spleen is at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, at least about7, at least about 10, at least about 12, at least about 14, at leastabout 21, at least about 28, at least about 35, at least about 42, atleast about 49, or at least about 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanTlast for spleen is at most about 1, at most about 2, at most about 3,at most about 4, at most about 5, at most about 6, at most about 7, atmost about 10, at most about 12, at most about 14, at most about 21, atmost about 28, at most about 35, at most about 42, at most about 49, atmost about 100 days, at most about 200, or at most about 300 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanTlast for spleen is about 1 to about 300, about 2 to about 100, about 5to about 50, about 10 to about 30, about 1 to about 20, about 20 toabout 40, about 40 to about 60, about 60 to about 80, or about 80 toabout 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(1/2) for spleen is at least 6, at least 24, at least 48, at least100, at least 150, at least 200, at least 250, at least 300, at least400, at least 500, or at least 1000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the mean T½for spleen is at most 24, at most 48, at most 100, at most 200, at most300, at most 400, at most 500, at most 600, at most 750, at most 1000,or at most 5000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the mean T½for spleen is about 24 to about 20000, about 50 to about 15000, about 50to about 1000, about 100 to about 500, about 100 to about 300, about 100to about 200, about 200 to about 300, about 300 to about 400, about 400to about 500, or about 3000 to about 5000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(max) of the compound in spleen is atleast 0.5, at least 1, at least 2, at least 4, at least 8, at least 12,at least 16, at least 20, at least 24, at least 28, at least 32, atleast 36, at least 40, at least 44, or at least 48 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(max) of the compound in spleen is atmost 6, at most 12, at most 16, at most 20, at most 24, at most 28, atmost 32, at most 36, at most 40, at most 44, at most 48, at most 72, atmost 96, or at most 120 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(max) of the compound in spleen isabout 0.5 to about 120, about 1 to about 24, about 1 to about 36, about1 to about 48, about 2 to about 36, about 6 to about 24, about 1 toabout 6, about 6 to about 12, about 12 to about 24, about 12 to about18, about 18 to about 48, about 18 to about 36, or about 24 to about 48hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean C_(max) of the compound in spleen is atleast about 1, at least about 50, at least about 100, at least about250, at least about 500, at least about 750, at least about 1250, atleast about 1500, at least about 2000, at least about 2500, at leastabout 3000, at least about 5000, or at least about 10000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Cmax of the compound in spleen is atmost about 1000, at most about 1500, at most about 2000, at most about2500, at most about 3000, at most about 4000, at most about 5000, or atmost about 10000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Cmax of the compound in spleen is about100 to about 50000, about 200 to about 5000, about 300 to about 4000,about 500 to about 3000, about 500 to about 1000, about 1000 to about2000, about 1000 to about 3000, or about 2000 to about 4000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean AUC_(last) of the compound for spleenis at least about 0.01×10{circumflex over ( )}6, at least about0.05×10{circumflex over ( )}6, at least about 0.08×10{circumflex over( )}6, at least about 0.1×10{circumflex over ( )}6, at least about0.2×10{circumflex over ( )}6, at least about 0.3×10{circumflex over( )}6, at least about 0.4×10{circumflex over ( )}6, at least about0.5×10{circumflex over ( )}6, at least about 0.75×10{circumflex over( )}6, at least about 1×10{circumflex over ( )}6, at least about3×10{circumflex over ( )}6, or at least about 5×10{circumflex over ( )}6ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean AUClast of the compound for spleen isat most about 0.08×10{circumflex over ( )}6, at most about0.1×10{circumflex over ( )}6, at most about 0.2×10{circumflex over( )}6, at most about 0.3×10{circumflex over ( )}6, at most about0.4×10{circumflex over ( )}6, at most about 0.5×10{circumflex over( )}6, at most about 0.75×10{circumflex over ( )}6, at most about1×10{circumflex over ( )}6, at most about 1.5×10{circumflex over ( )}6,at most about 2×10{circumflex over ( )}6, or at most about5×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean AUClast of the compound for spleen isabout 0.01×10{circumflex over ( )}6 to about 5×10{circumflex over ( )}6,about 0.05×10{circumflex over ( )}6 to about 3×10{circumflex over ( )}6,about 0.075×10{circumflex over ( )}6 to about 2×10{circumflex over( )}6, about 0.09×10{circumflex over ( )}6 to about 1.1×10{circumflexover ( )}6, about 0.1×10{circumflex over ( )}6 to about0.8×10{circumflex over ( )}6, about 0.1×10{circumflex over ( )}6 toabout 0.5×10{circumflex over ( )}6, about 0.1×10{circumflex over ( )}6to about 0.3×10{circumflex over ( )}6, about 0.3×10{circumflex over( )}6 to about 1×10{circumflex over ( )}6, about 0.5×10{circumflex over( )}6 to about 1×10{circumflex over ( )}6, about 0.1×10{circumflex over( )}6 to about 0.2×10{circumflex over ( )}6, about 0.2×10{circumflexover ( )}6 to about 0.3×10{circumflex over ( )}6, or about0.3×10{circumflex over ( )}6 to about 0.5×10{circumflex over ( )}6ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Tlast for spleen is at least about 1,at least about 2, at least about 3, at least about 4, at least about 5,at least about 6, at least about 7, at least about 10, at least about12, at least about 14, at least about 21, at least about 28, at leastabout 35, at least about 42, at least about 49, or at least about 100days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Tlast for spleen is at most about 1, atmost about 2, at most about 3, at most about 4, at most about 5, at mostabout 6, at most about 7, at most about 10, at most about 12, at mostabout 14, at most about 21, at most about 28, at most about 35, at mostabout 42, at most about 49, at most about 100 days, at most about 200,or at most about 300 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Tlast for spleen is about 1 to about300, about 2 to about 100, about 5 to about 50, about 10 to about 30,about 1 to about 20, about 20 to about 40, about 40 to about 60, about60 to about 80, or about 80 to about 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(1/2) for spleen is at least 6, atleast 24, at least 48, at least 100, at least 150, at least 200, atleast 250, at least 300, at least 400, at least 500, or at least 1000hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T½ for spleen is at most 24, at most48, at most 100, at most 200, at most 300, at most 400, at most 500, atmost 600, at most 750, at most 1000, or at most 5000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T½ for spleen is about 24 to about20000, about 50 to about 15000, about 50 to about 1000, about 100 toabout 500, about 100 to about 300, about 100 to about 200, about 200 toabout 300, about 300 to about 400, about 400 to about 500, or about 3000to about 5000 hours.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 4 hour at 0.3 mg/kg,after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 8 hour at 0.3 mg/kg,after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 24 hour at 0.6 mg/kg,after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 24 hour at 0.6 mg/kg,after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 48 hour at 1 mg/kg,after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 48 hour at 1 mg/kg,after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 756 ng/mL at 0.3mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 586 ng/mL at 0.3mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 1530 ng/mL at 0.6mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 984 ng/mL at 0.6mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 2940 ng/mL at 1mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 2270 ng/mL at 1mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 48300 ng·h/mLat 0.3 mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 96500 ng·h/mLat 0.3 mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 166000 ng·h/mLat 0.6 mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 98300 ng·h/mLat 0.6 mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 1010000ng·h/mL at 1 mg/kg, after administration of a single dose to a malesubject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 449000 ng·h/mLat 1 mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(last) ofthe compound is within 70% to 130% of a T_(last) of 144 hours at 0.3mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(last) ofthe compound is within 70% to 130% of a T_(last) of 648 hours at 0.3mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(last) ofthe compound is within 70% to 130% of a T_(last) of 312 hours at 0.6mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(last) ofthe compound is within 70% to 130% of a T_(last) of 648 hours at 0.6mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(last) ofthe compound is within 70% to 130% of a T_(last) of 648 hours at 1mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(last) ofthe compound is within 70% to 130% of a T_(last) of 312 hours at 1mg/kg, after administration of a single dose to a female subject.

Heart Pharmacokinetics

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(max) of the compound in heart is at least 0.5, at least 1, at least2, at least 4, at least 8, at least 12, at least 16, at least 20, atleast 24, at least 28, at least 32, at least 36, at least 40, at least44, or at least 48 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(max) of the compound in heart is at most 6, at most 12, at most 16,at most 20, at most 24, at most 28, at most 32, at most 36, at most 40,at most 44, at most 48, at most 72, at most 96, or at most 120 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(max) of the compound in heart is about 0.5 to about 120, about 1 toabout 24, about 1 to about 36, about 1 to about 48, about 2 to about 36,about 6 to about 24, about 1 to about 6, about 6 to about 12, about 12to about 24, about 12 to about 18, about 18 to about 48, about 18 toabout 36, or about 24 to about 48 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanC_(max) of the compound in heart is at least about 1, at least about 50,at least about 100, at least about 250, at least about 500, at leastabout 750, at least about 1250, at least about 1500, at least about2000, at least about 2500, at least about 3000, at least about 5000, orat least about 10000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanCmax of the compound in heart is at most about 750, at most about 1000,at most about 1500, at most about 2000, at most about 2500, at mostabout 3000, at most about 4000, at most about 5000, or at most about10000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanCmax of the compound in heart is about 100 to about 50000, about 200 toabout 5000, about 300 to about 4000, about 300 to about 3000, about 300to about 1000, about 100 to about 2000, about 1000 to about 3000, orabout 2000 to about 4000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanAUC_(last) of the compound for heart is at least about0.01×10{circumflex over ( )}6, at least about 0.05×10{circumflex over( )}6, at least about 0.08×10{circumflex over ( )}6, at least about0.1×10{circumflex over ( )}6, at least about 0.2×10{circumflex over( )}6, at least about 0.3×10{circumflex over ( )}6, at least about0.4×10{circumflex over ( )}6, at least about 0.5×10{circumflex over( )}6, at least about 0.75×10{circumflex over ( )}6, at least about1×10{circumflex over ( )}6, at least about 3×10{circumflex over ( )}6,or at least about 5×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanAUC_(last) of the compound for heart is at most about 0.08×10{circumflexover ( )}6, at most about 0.1×10{circumflex over ( )}6, at most about0.2×10{circumflex over ( )}6, at most about 0.3×10{circumflex over( )}6, at most about 0.4×10{circumflex over ( )}6, at most about0.5×10{circumflex over ( )}6, at most about 0.75×10{circumflex over( )}6, at most about 1×10{circumflex over ( )}6, at most about1.5×10{circumflex over ( )}6, at most about 2×10{circumflex over ( )}6,or at most about 5×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanAUC_(last) of the compound for heart is about 0.01×10{circumflex over( )}6 to about 5×10{circumflex over ( )}6, about 0.05×10{circumflex over( )}6 to about 3×10{circumflex over ( )}6, about 0.075×10{circumflexover ( )}6 to about 2×10{circumflex over ( )}6, about 0.09×10{circumflexover ( )}6 to about 1.1×10{circumflex over ( )}6, about0.1×10{circumflex over ( )}6 to about 0.8×10{circumflex over ( )}6,about 0.1×10{circumflex over ( )}6 to about 0.5×10{circumflex over( )}6, about 0.1×10{circumflex over ( )}6 to about 0.3×10{circumflexover ( )}6, about 0.3×10{circumflex over ( )}6 to about 1×10{circumflexover ( )}6, about 0.5×10{circumflex over ( )}6 to about 1×10{circumflexover ( )}6, about 0.1×10{circumflex over ( )}6 to about0.2×10{circumflex over ( )}6, about 0.2×10{circumflex over ( )}6 toabout 0.3×10{circumflex over ( )}6, or about 0.3×10{circumflex over( )}6 to about 0.5×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanTlast for heart is at least about 1, at least about 2, at least about 3,at least about 4, at least about 5, at least about 6, at least about 7,at least about 10, at least about 12, at least about 14, at least about21, at least about 28, at least about 35, at least about 42, at leastabout 49, or at least about 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanTlast for heart is at most about 1, at most about 2, at most about 3, atmost about 4, at most about 5, at most about 6, at most about 7, at mostabout 10, at most about 12, at most about 14, at most about 21, at mostabout 28, at most about 35, at most about 42, at most about 49, at mostabout 100 days, at most about 200, or at most about 300 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanTlast for heart is about 1 to about 300, about 2 to about 100, about 5to about 50, about 10 to about 30, about 1 to about 20, about 20 toabout 40, about 40 to about 60, about 60 to about 80, or about 80 toabout 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the meanT_(1/2) for heart is at least 6, at least 24, at least 48, at least 100,at least 150, at least 200, at least 250, at least 300, at least 400, atleast 500, or at least 1000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the mean T½for heart is at most 24, at most 48, at most 100, at most 200, at most300, at most 400, at most 500, at most 600, at most 750, at most 1000,or at most 5000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.01 mg/kg to about5 mg/kg (for example, about 0.1 to about 2 mg/kg, about 0.2 to about 1.5mg/kg, about 0.3 to about 1.2 mg/kg, about 0.3 to about 1 mg/kg, about0.3 to about 0.6 mg/kg, or about 0.6 to about 1 mg/kg), then the mean T½for heart is about 24 to about 20000, about 50 to about 15000, about 50to about 1000, about 100 to about 500, about 100 to about 300, about 100to about 200, about 200 to about 300, about 300 to about 400, about 400to about 500, or about 3000 to about 5000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(max) of the compound in heart is atleast 0.5, at least 1, at least 2, at least 4, at least 8, at least 12,at least 16, at least 20, at least 24, at least 28, at least 32, atleast 36, at least 40, at least 44, or at least 48 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(max) of the compound in heart is atmost 6, at most 12, at most 16, at most 20, at most 24, at most 28, atmost 32, at most 36, at most 40, at most 44, at most 48, at most 72, atmost 96, or at most 120 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T_(max) of the compound in heart isabout 0.5 to about 120, about 1 to about 24, about 1 to about 36, about1 to about 48, about 2 to about 36, about 6 to about 24, about 1 toabout 6, about 6 to about 12, about 12 to about 24, about 12 to about18, about 18 to about 48, about 18 to about 36, or about 24 to about 48hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean C_(max) of the compound in heart is atleast about 1, at least about 50, at least about 100, at least about250, at least about 500, at least about 750, at least about 1250, atleast about 1500, at least about 2000, at least about 2500, at leastabout 3000, at least about 5000, or at least about 10000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean C_(max) of the compound in heart is atmost about 1000, at most about 1500, at most about 2000, at most about2500, at most about 3000, at most about 4000, at most about 5000, or atmost about 10000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean C_(max) of the compound in heart isabout 100 to about 50000, about 200 to about 5000, about 300 to about4000, about 500 to about 3000, about 500 to about 1000, about 1000 toabout 2000, about 1000 to about 3000, or about 2000 to about 4000 ng/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean AUC_(last) of the compound for heart isat least about 0.01×10{circumflex over ( )}6, at least about0.05×10{circumflex over ( )}6, at least about 0.08×10{circumflex over( )}6, at least about 0.1×10{circumflex over ( )}6, at least about0.2×10{circumflex over ( )}6, at least about 0.3×10{circumflex over( )}6, at least about 0.4×10{circumflex over ( )}6, at least about0.5×10{circumflex over ( )}6, at least about 0.75×10{circumflex over( )}6, at least about 1×10{circumflex over ( )}6, at least about3×10{circumflex over ( )}6, or at least about 5×10{circumflex over ( )}6ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean AUC_(last) of the compound for heart isat most about 0.08×10{circumflex over ( )}6, at most about0.1×10{circumflex over ( )}6, at most about 0.2×10{circumflex over( )}6, at most about 0.3×10{circumflex over ( )}6, at most about0.4×10{circumflex over ( )}6, at most about 0.5×10{circumflex over( )}6, at most about 0.75×10{circumflex over ( )}6, at most about1×10{circumflex over ( )}6, at most about 1.5×10{circumflex over ( )}6,at most about 2×10{circumflex over ( )}6, or at most about5×10{circumflex over ( )}6 ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean AUC_(last) of the compound for heart isabout 0.01×10{circumflex over ( )}6 to about 5×10{circumflex over ( )}6,about 0.05×10{circumflex over ( )}6 to about 3×10{circumflex over ( )}6,about 0.075×10{circumflex over ( )}6 to about 2×10{circumflex over( )}6, about 0.09×10{circumflex over ( )}6 to about 1.1×10{circumflexover ( )}6, about 0.1×10{circumflex over ( )}6 to about0.8×10{circumflex over ( )}6, about 0.1×10{circumflex over ( )}6 toabout 0.5×10{circumflex over ( )}6, about 0.1×10{circumflex over ( )}6to about 0.3×10{circumflex over ( )}6, about 0.3×10{circumflex over( )}6 to about 1×10{circumflex over ( )}6, about 0.5×10{circumflex over( )}6 to about 1×10{circumflex over ( )}6, about 0.1×10{circumflex over( )}6 to about 0.2×10{circumflex over ( )}6, about 0.2×10{circumflexover ( )}6 to about 0.3×10{circumflex over ( )}6, or about0.3×10{circumflex over ( )}6 to about 0.5×10{circumflex over ( )}6ng·h/mL.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Tlast for heart is at least about 1, atleast about 2, at least about 3, at least about 4, at least about 5, atleast about 6, at least about 7, at least about 10, at least about 12,at least about 14, at least about 21, at least about 28, at least about35, at least about 42, at least about 49, or at least about 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Tlast for heart is at most about 1, atmost about 2, at most about 3, at most about 4, at most about 5, at mostabout 6, at most about 7, at most about 10, at most about 12, at mostabout 14, at most about 21, at most about 28, at most about 35, at mostabout 42, at most about 49, at most about 100 days, at most about 200,or at most about 300 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean Tlast for heart is about 1 to about300, about 2 to about 100, about 5 to about 50, about 10 to about 30,about 1 to about 20, about 20 to about 40, about 40 to about 60, about60 to about 80, or about 80 to about 100 days.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T½ for heart is at least 6, at least24, at least 48, at least 100, at least 150, at least 200, at least 250,at least 300, at least 400, at least 500, or at least 1000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T½ for heart is at most 24, at most 48,at most 100, at most 200, at most 300, at most 400, at most 500, at most600, at most 750, at most 1000, or at most 5000 hours.

In some embodiments, if a pharmacokinetic study is conducted, whereinthe pharmacokinetic study comprises administering (e.g., viaintracerebroventricular administration) a compound of the disclosure toa study patient or subject at a dose amount of about 0.001 mg/kg toabout 0.5 mg/kg (for example, about 0.008 to about 0.163 mg/kg, about0.016 to about 0.125 mg/kg, about 0.025 to about 0.1 mg/kg, about 0.025to about 0.08 mg/kg, about 0.025 to about 0.05 mg/kg, or about 0.05 toabout 0.08 mg/kg), then the mean T½ for heart is about 24 to about20000, about 50 to about 15000, about 50 to about 1000, about 100 toabout 500, about 100 to about 300, about 100 to about 200, about 200 toabout 300, about 300 to about 400, about 400 to about 500, or about 3000to about 5000 hours.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 48 hours at 0.3mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(max) ofthe compound is within 70% to 130% of a T_(max) of 24 hours at 0.3mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 416 ng/mL at 0.3mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean C_(max) ofthe compound is within 70% to 130% of a C_(max) of 729 ng/mL at 0.3mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 160000 ng·h/mLat 0.3 mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean AUC_(last)of the compound is within 70% to 130% of an AUC_(last) of 184000 ng·h/mLat 0.3 mg/kg, after administration of a single dose to a female subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(last) ofthe compound is within 70% to 130% of a T_(last) of 648 hours at 0.3mg/kg, after administration of a single dose to a male subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intracerebroventricular delivery of a compoundcomprising a peptide nucleic acid sequence, wherein the mean T_(last) ofthe compound is within 70% to 130% of a T_(last) of 648 hours at 0.3mg/kg, after administration of a single dose to a female subject.

Biodistribution.

Biodistribution of compounds of the disclosure can be evaluated viamethods that can directly or indirectly detect the presence of compoundin tissue. For example, tissue of a subject administered a compound ofthe disclosure can be evaluated by mass spectroscopic methods, such astandem mass spectrometry, whereby presence of compound in tissue samplescan be evaluated on the basis of intensity of a signal corresponding tothe mass of ionized compound or compound fragments. Other methodssuitable for determination of biodistribution include administration ofa radiolabeled analogue of the compound, and evaluation of theradiographic signature of the analogue in the subject via imagingtechniques such as autoradiography, positron emission tomography, orsingle-photon emission computed tomography.

In some embodiments, biodistribution of a radiolabeled analogue isevaluated via quantitative whole body autoradiography (QWBA). Inquantitative whole body autoradiography, an animal subject isadministered the radiolabeled analogue, euthanized at a specifiedtimepoint subsequent to administration, frozen, and suspended inembedding media such as aqueous sodium carboxymethyl cellulose. Thesuspended carcass can be sectioned in a cryomacrotome, and the resultingsections can be mounted on an adhesive support and placed on an imagingplate sensitive to the specific radioisotope used in the radiolabeledanalogue, such as carbon-14. The exposed imaging plates can then beconverted to electronic form using a phosphor imager system, andselected areas of the image file can be electronically integrated toprovide concentrations of analogue expressed in ng-equivalents ofcompound per gram of tissue (ng-eq/g).

The QWBA assay can be repeated on multiple animal subjects, or selectedparameters of the assay can be varied from animal to animal, such asdosage, route of administration, or time from compound administration toeuthanization. For example, a QWBA can comprise three components, whereone animal is euthanized four hours after administration of aradiolabeled analogue, a second animal is euthanized 12 hours afteradministration of the radiolabeled analogue, and a third animal iseuthanized 7 days after administration of the radiolabeled analogue.Concentrations of the analogue in various tissues of each animal canthen be determined and compared across each animal, thereby providinginsight into the timecourse of biodistribution of the radiolabeledanalogue and compound. In each study component, urine and feces of eachsubject animal can be collected and evaluated for radiolabeled analoguecontent.

Upon systemic administration, a compound of the disclosure, or aradiolabeled analogue thereof can exhibit distribution into certaintissues, for example, as determined by quantitative whole-bodyautoradiography (QWBA).

In some embodiments, if an assay is conducted (e.g., tissue distributionstudy), wherein the assay comprises administering (e.g., via single doseintravenous administration) a radiolabeled analogue of a compound of thedisclosure (e.g., [^(14C)]-Compound 1) to a study patient (e.g., amonkey) or subject at a dose amount of about 5 mg/kg (for example, about0.05 to about 500 mg/kg, about 0.5 to about 50 mg/kg, about 1 to about10 mg/kg, about 2 to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4to about 6 mg/kg), then the compound can be distributed to, for example,kidney cortex, joints, cartilage, liver, salivary glands, bone surface,pancreas, hair follicles, large intestine mucosa, aortic wall, smallintestine mucosa, adrenal gland, stomach mucosa, spleen, bone marrow,lymph nodes, thymus, brain, cerebellum, olfactory bulb, thalamus,caudate putamen, cerebral cortex, substantia nigra, lateral ventricle,choroid plexus, or a combination thereof, for example, as determined byquantitative whole-body autoradiography (QWBA). In some embodiments, thecompound is distributed in the tissue for at least 6, at least 24, atleast 48, at least 100, at least 125, at least 150, at least 200, atleast 250, at least 300, at least 400, at least 500, or at least 1000hours.

In some embodiments, if in a study or assay (e.g., tissue distributionstudy), the compound is administered (e.g., via single dose intravenousadministration) to a study patient or subject at a dose amount of about5 mg/kg (for example, about 0.05 to about 500 mg/kg, about 0.5 to about50 mg/kg, about 1 to about 10 mg/kg, about 2 to about 8 mg/kg, about 3to about 7 mg/kg, or about 4 to about 6 mg/kg), then the compound can bedistributed to, for example, Adrenal gland cortex, Adrenal glandmedulla, Aortic wall, Bone inner, Bone surface, Bone marrow, Braincaudate, Brain cerebellum, Brain cortex, Brain lateral ventricle, Brainolfactory bulb, Brain putamen, Brain substantia nigra, Brain thalamus,Brown fat, Eye uveal tract (choroid+RPE), Hair follicles, Heart blood,Heart myocardium, Joints (cartilage), Kidney cortex, Kidney medulla,Large intestine content, Large intestine mucosa, Large intestine wall,Liver, Lung, Lymph nodes, Pancreas, Pituitary gland, Salivaryglands—Parotid, Salivary glands—other, Skeletal muscle, Skin, Smallintestine content, Small intestine mucosa, Small intestine wall, Spinalcord, Spleen, Stomach content, Stomach mucosa, Stomach wall, Testis,Thymus, Thyroid gland, Urinary Bladder, Urine, White fat, Whole blood,or a combination thereof, for example, as determined by quantitativewhole-body autoradiography (QWBA). In some embodiments, the compound isdistributed in the tissue for at least 6, at least 24, at least 48, atleast 100, at least 125, at least 150, at least 200, at least 250, atleast 300, at least 400, at least 500, or at least 1000 hours.

In some embodiments, the disclosure provides a compound comprising apeptide nucleic acid sequence and a cell permeabilizing group attachedto the peptide nucleic acid sequence. In an assay of a retention of thecompound in a brain of a subject following administration of thecompound to the subject, an amount of the compound within the brain atseven, eight, nine, or ten days subsequent to the administration can beequivalent to at least 80% of an amount of the compound within the brainat 4 hours subsequent to the administration. In some embodiments, theamount of the compound within the brain at seven, eight, nine, or tendays subsequent to the administration is equivalent to at least 100% ofthe amount of the compound within the brain at 4 hours subsequent to theadministration. In some embodiments, the amount of the compound withinthe brain at seven, eight, nine, or ten days subsequent to theadministration is equivalent to at least 150% of the amount of thecompound within the brain at 4 hours subsequent to the administration.In some embodiments, the amount of the compound within the brain atseven, eight, nine, or ten days subsequent to the administration isequivalent to at least 200% of the amount of the compound within thebrain at 4 hours subsequent to the administration.

In some embodiments, the disclosure provides a compound comprising apeptide nucleic acid sequence and a cell permeabilizing group attachedto the peptide nucleic acid sequence. In an assay of a retention of thecompound in a brain of a subject following administration of thecompound to the subject, least 80% of the compound present in the brainat 4 hours subsequent to the administration can remain in the brain forat least 7 days following the administration. In some embodiments, theassay is a quantitative whole body autoradiography assay.

In some embodiments, the disclosure provides a compound comprising apeptide nucleic acid sequence and a cell permeabilizing group attachedto the peptide nucleic acid sequence. In an assay of a retention of thecompound in a skeletal muscle of a subject following administration ofthe compound to the subject, an amount of the compound within theskeletal muscle at seven, eight, nine, or ten days subsequent to theadministration may be equivalent to at least 40% of an amount of thecompound within the skeletal muscle at 4 hours subsequent to theadministration. In some embodiments, the amount of the compound withinthe skeletal muscle at seven, eight, nine, or ten days subsequent to theadministration is equivalent to at least 60% of the amount of thecompound within the skeletal muscle at 4 hours subsequent to theadministration. In some embodiments, the amount of the compound withinthe skeletal muscle at seven, eight, nine, or ten days subsequent to theadministration is equivalent to at least 80% of the amount of thecompound within the skeletal muscle at 4 hours subsequent to theadministration. In some embodiments, the amount of the compound withinthe skeletal muscle at seven, eight, nine, or ten days subsequent to theadministration is equivalent to at least 90% of the amount of thecompound within the skeletal muscle at 4 hours subsequent to theadministration. In some embodiments, the assay is a quantitative wholebody autoradiography assay.

In some embodiments, the disclosure provides a compound comprising apeptide nucleic acid sequence and a cell permeabilizing group attachedto the peptide nucleic acid sequence. In an assay of a retention of thecompound in skeletal muscle of a subject following administration of thecompound to the subject, least 80% of the compound present in theskeletal muscle at 4 hours subsequent to the administration can remainin the skeletal muscle for at least 7 days following the administration.In some embodiments, the assay is a quantitative whole bodyautoradiography assay.

In some embodiments, a concentration of the compound in the brain of thesubject is at least about 100 nanograms of the compound per gram of wetbrain tissue, 200 nanograms of the compound per gram of wet braintissue, 300 nanograms of the compound per gram of wet brain tissue, 400nanograms of the compound per gram of wet brain tissue, 500 nanograms ofthe compound per gram of wet brain tissue, 600 nanograms of the compoundper gram of wet brain tissue, 700 nanograms of the compound per gram ofwet brain tissue, 800 nanograms of the compound per gram of wet braintissue, 900 nanograms of the compound per gram of wet brain tissue, or1000 nanograms of the compound per gram of wet brain tissue after about4 hours subsequent to the administering.

In some embodiments, a concentration of the compound in the brain of thesubject is at least about 100 nanograms of the compound per gram of wetbrain tissue, 200 nanograms of the compound per gram of wet braintissue, 300 nanograms of the compound per gram of wet brain tissue, 400nanograms of the compound per gram of wet brain tissue, 500 nanograms ofthe compound per gram of wet brain tissue, 600 nanograms of the compoundper gram of wet brain tissue, 700 nanograms of the compound per gram ofwet brain tissue, 800 nanograms of the compound per gram of wet braintissue, 900 nanograms of the compound per gram of wet brain tissue, or1000 nanograms of the compound per gram of wet brain tissue after about7 days subsequent to the administering.

In some embodiments, a concentration of the compound the brain of thesubject is at least about 200 nanograms of the compound per gram of wetbrain tissue after about 4 hours subsequent to the administering. Insome embodiments, a concentration of the compound in the brain of thesubject is at least about 200 nanograms of the compound per gram of wetbrain tissue after about 7 days subsequent to the administering. In someembodiments, a concentration of the compound in the brain of the subjectis at least about 100 nanomoles of the compound per liter after about 4hours subsequent to the administering. In some embodiments, aconcentration of the compound in the brain of the subject is at leastabout 100 nanomoles of the compound per liter after about 7 dayssubsequent to the administering.

Upon systemic administration, a compound of the disclosure or aradiolabeled analogue thereof can exhibit a low rate of excretion.

In some embodiments, if an assay is conducted (e.g., tissue distributionstudy), wherein the assay comprises administering (e.g., via single doseintravenous administration) a compound of the disclosure (e.g.,Compound 1) or a radiolabeled analogue thereof (e.g.,[^(14C)]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 5 mg/kg (for example, about 0.05 to about 500mg/kg, about 0.5 to about 50 mg/kg, about 1 to about 10 mg/kg, about 2to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4 to about 6mg/kg), then less than about 20%, less than about 15%, less than about10%, less than about 9%, less than about 8%, less than about 7%, lessthan about 6%, less than about 5%, less than about 4%, less than about3%, less than about 2.5%, less than about 2%, less than about 1.9%, lessthan about 1.8%, less than about 1.7%, less than about 1.6%, less thanabout 1.5%, less than about 1.4%, less than about 1.3%, less than about1.2%, less than about 1.1%, or less than about 1% of the dose can berecovered from urine, for example, over about 168.

In some embodiments, if an assay is conducted (e.g., tissue distributionstudy), wherein the assay comprises administering (e.g., via single doseintravenous administration) a compound of the disclosure (e.g.,Compound 1) or a radiolabeled analogue thereof (e.g., [¹⁴C]-Compound 1)to a study patient (e.g., a monkey) or subject at a dose amount of about5 mg/kg (for example, about 0.05 to about 500 mg/kg, about 0.5 to about50 mg/kg, about 1 to about 10 mg/kg, about 2 to about 8 mg/kg, about 3to about 7 mg/kg, or about 4 to about 6 mg/kg), then less than about20%, less than about 15%, less than about 10%, less than about 9%, lessthan about 8%, less than about 7%, less than about 6%, less than about5%, less than about 4%, less than about 3%, less than about 2.5%, lessthan about 2%, less than about 1.9%, less than about 1.8%, less thanabout 1.7%, less than about 1.6%, less than about 1.5%, less than about1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%,or less than about 1% of the dose can be recovered from feces, forexample, over about 168 hours.

In some embodiments, if an assay is conducted (e.g., tissue distributionstudy), wherein the assay comprises administering (e.g., via single doseintravenous administration) a compound of the disclosure (e.g.,Compound 1) or a radiolabeled analogue thereof (e.g.,[^(14C)]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 5 mg/kg (for example, about 0.05 to about 500mg/kg, about 0.5 to about 50 mg/kg, about 1 to about 10 mg/kg, about 2to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4 to about 6mg/kg), then less than about 20%, less than about 15%, less than about10%, less than about 9%, less than about 8%, less than about 7%, lessthan about 6%, less than about 5%, less than about 4%, less than about3%, less than about 2.5%, less than about 2%, less than about 1.9%, lessthan about 1.8%, less than about 1.7%, less than about 1.6%, less thanabout 1.5%, less than about 1.4%, less than about 1.3%, less than about1.2%, less than about 1.1%, or less than about 1% of the dose can beexcreted, for example, over about 168 hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 5 mg/kg (for example, about 0.05 to about 500mg/kg, about 0.5 to about 50 mg/kg, about 1 to about 10 mg/kg, about 2to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4 to about 6mg/kg), then the mean T_(1/2) for plasma is at least 6, at least 24, atleast 48, at least 100, at least 125, at least 150, at least 200, atleast 250, at least 300, at least 400, at least 500, or at least 1000hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 5 mg/kg (for example, about 0.05 to about 500mg/kg, about 0.5 to about 50 mg/kg, about 1 to about 10 mg/kg, about 2to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4 to about 6mg/kg), then a mean T_(1/2) for plasma is at most 24, at most 48, atmost 100, at most 200, at most 300, at most 400, at most 500, at most600, at most 750, at most 1000, or at most 5000 hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 5 mg/kg (for example, about 0.05 to about 500mg/kg, about 0.5 to about 50 mg/kg, about 1 to about 10 mg/kg, about 2to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4 to about 6mg/kg), then a mean T_(1/2) for plasma is about 24 to about 20000, about50 to about 15000, about 50 to about 1000, about 100 to about 500, about100 to about 300, about 100 to about 200, about 200 to about 300, about300 to about 400, about 400 to about 500, or about 3000 to about 5000hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g., [¹⁴C]-Compound 1) to a study patient(e.g., a monkey) or subject at a dose amount of about 5 mg/kg (forexample, about 0.05 to about 500 mg/kg, about 0.5 to about 50 mg/kg,about 1 to about 10 mg/kg, about 2 to about 8 mg/kg, about 3 to about 7mg/kg, or about 4 to about 6 mg/kg), then the mean C_(max) of thecompound in plasma is at least about 1, at least about 1000, at leastabout 2000, at least about 3000, at least about 4000, at least about5000, at least about 6000, at least about 7000, at least about 8000, atleast about 9000, at least about 10000, at least about 12000, at leastabout 14000, at least about 16000, at least about 18000, at least about20000, at least about 22000, or at least about 25000 ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 5 mg/kg (for example, about 0.05 to about 500mg/kg, about 0.5 to about 50 mg/kg, about 1 to about 10 mg/kg, about 2to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4 to about 6mg/kg), then the mean C_(max) of the compound in plasma is at most about3000, at most about 4000, at most about 5000, at most about 6000, atmost about 7000, at most about 8000, at most about 9000, at most about10000, at most about 12000, at most about 14000, at most about 16000, atmost about 18000, at most about 20000, at most about 22000, at mostabout 25000, at most about 30000 or at most about 50000 ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g., [¹⁴C]-Compound 1) to a study patient(e.g., a monkey) or subject at a dose amount of about 5 mg/kg (forexample, about 0.05 to about 500 mg/kg, about 0.5 to about 50 mg/kg,about 1 to about 10 mg/kg, about 2 to about 8 mg/kg, about 3 to about 7mg/kg, or about 4 to about 6 mg/kg), then the mean C_(max) of thecompound in plasma is about 1000 to about 50000, about 2000 to about25000, about 3000 to about 22000, about 2000 to about 5000, about 5000to about 15000, or about 10000 to about 25000 ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 1.6 mg/kg (for example, about 0.015 to about 160mg/kg, about 0.16 to about 16 mg/kg, about 0.3 to about 3.2 mg/kg, about0.65 to about 2.6 mg/kg, about 1 to about 2.3 mg/kg, or about 1.3 toabout 2 mg/kg), then the mean T_(1/2) for plasma is at least 6, at least24, at least 48, at least 100, at least 125, at least 150, at least 200,at least 250, at least 300, at least 400, at least 500, or at least 1000hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 1.6 mg/kg (for example, about 0.015 to about 160mg/kg, about 0.16 to about 16 mg/kg, about 0.3 to about 3.2 mg/kg, about0.65 to about 2.6 mg/kg, about 1 to about 2.3 mg/kg, or about 1.3 toabout 2 mg/kg), then a mean T_(1/2) for plasma is at most 24, at most48, at most 100, at most 200, at most 300, at most 400, at most 500, atmost 600, at most 750, at most 1000, or at most 5000 hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 1.6 mg/kg (for example, about 0.015 to about 160mg/kg, about 0.16 to about 16 mg/kg, about 0.3 to about 3.2 mg/kg, about0.65 to about 2.6 mg/kg, about 1 to about 2.3 mg/kg, or about 1.3 toabout 2 mg/kg), then a mean T_(1/2) for plasma is about 24 to about20000, about 50 to about 15000, about 50 to about 1000, about 100 toabout 500, about 100 to about 300, about 100 to about 200, about 200 toabout 300, about 300 to about 400, about 400 to about 500, or about 3000to about 5000 hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g., [¹⁴C]-Compound 1) to a study patient(e.g., a monkey) or subject at a dose amount of about 1.6 mg/kg (forexample, about 0.015 to about 160 mg/kg, about 0.16 to about 16 mg/kg,about 0.3 to about 3.2 mg/kg, about 0.65 to about 2.6 mg/kg, about 1 toabout 2.3 mg/kg, or about 1.3 to about 2 mg/kg), then the mean C_(max)of the compound in plasma is at least about 1, at least about 1000, atleast about 2000, at least about 3000, at least about 4000, at leastabout 5000, at least about 6000, at least about 7000, at least about8000, at least about 9000, at least about 10000, at least about 12000,at least about 14000, at least about 16000, at least about 18000, atleast about 20000, at least about 22000, or at least about 25000ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g., [^(14C)]-Compound 1) to a studypatient (e.g., a monkey) or subject at a dose amount of about 1.6 mg/kg(for example, about 0.015 to about 160 mg/kg, about 0.16 to about 16mg/kg, about 0.3 to about 3.2 mg/kg, about 0.65 to about 2.6 mg/kg,about 1 to about 2.3 mg/kg, or about 1.3 to about 2 mg/kg), then themean C_(max) of the compound in plasma is at most about 3000, at mostabout 4000, at most about 5000, at most about 6000, at most about 7000,at most about 8000, at most about 9000, at most about 10000, at mostabout 12000, at most about 14000, at most about 16000, at most about18000, at most about 20000, at most about 22000, at most about 25000, atmost about 30000 or at most about 50000 ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g., [¹⁴C]-Compound 1) to a study patient(e.g., a monkey) or subject at a dose amount of about 1.6 mg/kg (forexample, about 0.015 to about 160 mg/kg, about 0.16 to about 16 mg/kg,about 0.3 to about 3.2 mg/kg, about 0.65 to about 2.6 mg/kg, about 1 toabout 2.3 mg/kg, or about 1.3 to about 2 mg/kg), then the mean C_(max)of the compound in plasma is about 1000 to about 50000, about 2000 toabout 25000, about 3000 to about 22000, about 2000 to about 5000, about5000 to about 15000, or about 10000 to about 25000 ng-eq/g.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intravenous delivery of a compound comprising apeptide nucleic acid sequence or a radiolabeled analogue thereof,wherein the mean AUC_(0-t) of the compound is within 70% to 130% of anAUC_(0-t) of 205000 h·ng-eq/g in plasma, after administration of asingle dose to a subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intravenous delivery of a compound comprising apeptide nucleic acid sequence or a radiolabeled analogue thereof,wherein the mean C_(max) of the compound is within 70% to 130% of aC_(max) of 11500 ng-eq/g in plasma, after administration of a singledose to a subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intravenous delivery of a compound comprising apeptide nucleic acid sequence, wherein the mean t_(1/2) of the compoundis within 70% to 130% of a t_(1/2) of 141 hours in plasma, afteradministration of a single dose to a subject.

In some embodiments, the subject is a primate, a monkey, or a human.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 5 mg/kg (for example, about 0.05 to about 500mg/kg, about 0.5 to about 50 mg/kg, about 1 to about 10 mg/kg, about 2to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4 to about 6mg/kg), then the mean T_(1/2) for blood is at least 6, at least 24, atleast 48, at least 100, at least 125, at least 150, at least 200, atleast 250, at least 300, at least 400, at least 500, or at least 1000hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 5 mg/kg (for example, about 0.05 to about 500mg/kg, about 0.5 to about 50 mg/kg, about 1 to about 10 mg/kg, about 2to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4 to about 6mg/kg), then a mean T_(1/2) for blood is at most 24, at most 48, at most100, at most 200, at most 300, at most 400, at most 500, at most 600, atmost 750, at most 1000, or at most 5000 hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient (e.g., a monkey) or subject at adose amount of about 5 mg/kg (for example, about 0.05 to about 500mg/kg, about 0.5 to about 50 mg/kg, about 1 to about 10 mg/kg, about 2to about 8 mg/kg, about 3 to about 7 mg/kg, or about 4 to about 6mg/kg), then a mean T_(1/2) for blood is about 24 to about 20000, about50 to about 15000, about 50 to about 1000, about 100 to about 500, about100 to about 300, about 100 to about 200, about 200 to about 300, about300 to about 400, about 400 to about 500, or about 3000 to about 5000hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g., [¹⁴C]-Compound 1) to a study patient(e.g., monkey) or subject at a dose amount of about 5 mg/kg (forexample, about 0.05 to about 500 mg/kg, about 0.5 to about 50 mg/kg,about 1 to about 10 mg/kg, about 2 to about 8 mg/kg, about 3 to about 7mg/kg, or about 4 to about 6 mg/kg), then the mean C_(max) of thecompound in blood is at least about 1, at least about 1000, at leastabout 2000, at least about 3000, at least about 4000, at least about5000, at least about 6000, at least about 7000, at least about 8000, atleast about 9000, at least about 10000, at least about 12000, at leastabout 14000, at least about 16000, at least about 18000, at least about20000, at least about 22000, or at least about 25000 ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g [¹⁴C]-Compound 1) to a study patient(e.g., a monkey) or subject at a dose amount of about 5 mg/kg (forexample, about 0.05 to about 500 mg/kg, about 0.5 to about 50 mg/kg,about 1 to about 10 mg/kg, about 2 to about 8 mg/kg, about 3 to about 7mg/kg, or about 4 to about 6 mg/kg), then the mean C_(max) of thecompound in blood is at most about 3000, at most about 4000, at mostabout 5000, at most about 6000, at most about 7000, at most about 8000,at most about 9000, at most about 10000, at most about 12000, at mostabout 14000, at most about 16000, at most about 18000, at most about20000, at most about 22000, at most about 25000, at most about 30000 orat most about 50000 ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g [¹⁴C]-Compound 1) to a study patient(e.g., a monkey) or subject at a dose amount of about 5 mg/kg (forexample, about 0.05 to about 500 mg/kg, about 0.5 to about 50 mg/kg,about 1 to about 10 mg/kg, about 2 to about 8 mg/kg, about 3 to about 7mg/kg, or about 4 to about 6 mg/kg), then the mean C_(max) of thecompound in blood is about 1000 to about 50000, about 2000 to about25000, about 3000 to about 22000, about 2000 to about 5000, about 5000to about 15000, or about 10000 to about 25000 ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g[¹⁴C]-Compound 1) to a study patient or subject at a dose amount ofabout 1.6 mg/kg (for example, about 0.015 to about 160 mg/kg, about 0.16to about 16 mg/kg, about 0.3 to about 3.2 mg/kg, about 0.65 to about 2.6mg/kg, about 1 to about 2.3 mg/kg, or about 1.3 to about 2 mg/kg), thenthe mean T½ for blood is at least 6, at least 24, at least 48, at least100, at least 125, at least 150, at least 200, at least 250, at least300, at least 400, at least 500, or at least 1000 hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g[¹⁴C]-Compound 1) to a study patient or subject at a dose amount ofabout 1.6 mg/kg (for example, about 0.015 to about 160 mg/kg, about 0.16to about 16 mg/kg, about 0.3 to about 3.2 mg/kg, about 0.65 to about 2.6mg/kg, about 1 to about 2.3 mg/kg, or about 1.3 to about 2 mg/kg), thena mean T_(1/2) for blood is at most 24, at most 48, at most 100, at most200, at most 300, at most 400, at most 500, at most 600, at most 750, atmost 1000, or at most 5000 hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a compound of the disclosure(e.g., Compound 1) or a radiolabeled analogue thereof (e.g.,[¹⁴C]-Compound 1) to a study patient or subject at a dose amount ofabout 1.6 mg/kg (for example, about 0.015 to about 160 mg/kg, about 0.16to about 16 mg/kg, about 0.3 to about 3.2 mg/kg, about 0.65 to about 2.6mg/kg, about 1 to about 2.3 mg/kg, or about 1.3 to about 2 mg/kg), thena mean T_(1/2) for blood is about 24 to about 20000, about 50 to about15000, about 50 to about 1000, about 100 to about 500, about 100 toabout 300, about 100 to about 200, about 200 to about 300, about 300 toabout 400, about 400 to about 500, or about 3000 to about 5000 hours.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g., [¹⁴C]-Compound 1) to a study patientor subject at a dose amount of about 1.6 mg/kg (for example, about 0.015to about 160 mg/kg, about 0.16 to about 16 mg/kg, about 0.3 to about 3.2mg/kg, about 0.65 to about 2.6 mg/kg, about 1 to about 2.3 mg/kg, orabout 1.3 to about 2 mg/kg), then the mean C_(max) of the compound inblood is at least about 1, at least about 1000, at least about 2000, atleast about 3000, at least about 4000, at least about 5000, at leastabout 6000, at least about 7000, at least about 8000, at least about9000, at least about 10000, at least about 12000, at least about 14000,at least about 16000, at least about 18000, at least about 20000, atleast about 22000, or at least about 25000 ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g., [¹⁴C]-Compound 1) to a study patientor subject at a dose amount of about 1.6 mg/kg (for example, about 0.015to about 160 mg/kg, about 0.16 to about 16 mg/kg, about 0.3 to about 3.2mg/kg, about 0.65 to about 2.6 mg/kg, about 1 to about 2.3 mg/kg, orabout 1.3 to about 2 mg/kg), then the mean C_(max) of the compound inblood is at most about 3000, at most about 4000, at most about 5000, atmost about 6000, at most about 7000, at most about 8000, at most about9000, at most about 10000, at most about 12000, at most about 14000, atmost about 16000, at most about 18000, at most about 20000, at mostabout 22000, at most about 25000, at most about 30000 or at most about50000 ng-eq/g.

In some embodiments, if a study or assay is conducted (e.g., tissuedistribution study), wherein the assay comprises administering (e.g.,via single dose intravenous administration) a radiolabeled analogue of acompound of the disclosure (e.g., [^(14C)]-Compound 1) to a studypatient or subject at a dose amount of about 1.6 mg/kg (for example,about 0.015 to about 160 mg/kg, about 0.16 to about 16 mg/kg, about 0.3to about 3.2 mg/kg, about 0.65 to about 2.6 mg/kg, about 1 to about 2.3mg/kg, or about 1.3 to about 2 mg/kg), then the mean C_(max) of thecompound in blood is about 1000 to about 50000, about 2000 to about25000, about 3000 to about 22000, about 2000 to about 5000, about 5000to about 15000, or about 10000 to about 25000 ng-eq/g.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intravenous delivery of a compound comprising apeptide nucleic acid sequence, wherein the mean AUC_(0-t) of aradiolabeled analogue of the compound is within 70% to 130% of anAUC_(0-t) of 231000 h·ng-eq/g in blood, after administration of a singledose to a subject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intravenous delivery of a compound comprising apeptide nucleic acid sequence, wherein the mean C_(max) of aradiolabeled analogue of the compound is within 70% to 130% of a C_(max)of 7510 ng-eq/g in blood, after administration of a single dose to asubject.

In some embodiments, provided herein is a pharmaceutical composition inunit dose form for intravenous delivery of a compound comprising apeptide nucleic acid sequence, wherein the mean t_(1/2) of the compoundis within 70% to 130% of a t_(1/2) of 229 hours in blood, afteradministration of a single dose to a subject.

In some embodiments, the subject is a primate, a monkey, or a human.

In some embodiments, no more than 2% of the therapeutically-effectiveamount of the compound is excreted by the subject in urine over a 7 dayperiod subsequent to the administering. In some embodiments, no morethan 2% of the therapeutically-effective amount of the compound isexcreted by the subject in feces over a 7 day period subsequent to theadministering.

In some embodiments, a compound of the disclosure can achieve fullbiodistribution without lipid nanoparticles. For example, the backboneis covalently modified such that the compound exhibits cell permeabilitythat does not differ across cell types. For example, the backbone can becovalently modified with functional groups (e.g., guanidino groups) suchthat the compound exhibits cell permeability that does not significantlydiffer across cell types.

In some embodiments, if the compound, disclosed herein, is subjected toa plasma protein binding assay, then, in the plasma protein bindingassay, the plasma protein binding percentage is at least about 85% inone or more study patients. In some embodiments, the plasma proteinbinding percentage is at least about 85% in each of a human, mouse, dog,minipig, sheep, and/or monkey. In some embodiments, the plasma proteinbinding assay comprises performing the plasma protein binding assay on astudy patient. In some embodiments, at a concentration of about 1 μg/mL,the plasma protein binding percentage is at least about 95% in thehuman, mouse, dog, minipig, sheep, and/or monkey. In some embodiments,at the concentration of about 1 μg/mL, the plasma protein bindingpercentage is at least about 95% in each of the human, mouse, dog,minipig, sheep, and monkey.

In some embodiments, the plasma protein binding assay comprises spikingsingle aliquots of a study patient's plasma with a first solution of thecompound (e.g., 10 mg/mL of the first solution of the compound) toobtain at least a second solution of the compound (e.g., withconcentrations of about 1 μg/mL to about 50 μg/mL). In some embodiments,a separation technique (e.g., ultracentrifugation) is used on the atleast the second solution of the compound to separate a mixturecomprising the compounds that are bound to plasma proteins. In someembodiments, plasma protein binding percentage in the study patient'splasma can be determined using various techniques, disclosed herein,such as liquid chromatography-tandem mass spectrometry.

In some embodiments, the plasma protein binding assay comprisesperforming a human component of the plasma protein binding assay. Forexample, the human component of the plasma protein binding assaycomprises spiking single aliquots of human plasma with a first solutionof the compound (e.g., 10 mg/mL of the first solution of the compound)to obtain at least a second solution of the compound (e.g., withconcentrations of about 1 μg/mL to about 50 μg/mL). In some embodiments,a separation technique (e.g., ultracentrifugation) is used on the atleast the second solution of the compound to separate a mixturecomprising the compounds that are bound to plasma proteins. In someembodiments, a plasma protein binding percentage in the human plasma canbe determine using various techniques, disclosed herein, such as liquidchromatography-tandem mass spectrometry. In some embodiments, a mousecomponent of the plasma protein binding assay is performed. In someembodiments, the mouse component of the plasma protein binding assaydiffers from the human component of the plasma protein binding assayonly in that mouse plasma is used instead of the human plasma. In someembodiments, a dog component of the plasma protein binding assay isperformed. In some embodiments, the dog component of the plasma proteinbinding assay differs from the human component of the plasma proteinbinding assay only in that dog plasma is used instead of the humanplasma. In some embodiments, a minipig component of the plasma proteinbinding assay is performed. In some embodiments, the minipig componentof the plasma protein binding assay differs from the human component ofthe plasma protein binding assay only in that minipig plasma is usedinstead of the human plasma. In some embodiments, a sheep component ofthe plasma protein binding assay is performed. In some embodiments, thesheep component of the plasma protein binding assay differs from thehuman component of the plasma protein binding assay only in that sheepplasma is used instead of the human plasma. In some embodiments, amonkey component of the plasma protein binding assay is performed. Insome embodiments, the monkey component of the plasma protein bindingassay differs from the human component of the plasma protein bindingassay only in that monkey plasma is used instead of the human plasma.

In some embodiments, in a plasma binding protein assay (e.g., in vitroplasma binding protein assay), if the compound is assessed in plasmafrom a host species, (e.g., spiked into pooled plasma at a concentrationof about 0.01, about 0.1, about 0.5, about 1, about 5, about 10, about50, about 100, about 500, or about 1000 μg/mL), then the % binding ofthe compound to plasma proteins (PPB) is about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%,about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%,about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%,about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about98%, about 98.5%, about 99%, about 99.1%, about 99.1%, about 99.3%,about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about99.9%, about 99.95%, or about 99.99%. The host species can be, forexample, mouse (e.g., C57BL6), dog (e.g., beagle), minipig (e.g.,Gottingen minipig), sheep, monkey (e.g., Cynomolgus monkey), human, orany suitable animal disclosed herein.

In some embodiments, in a plasma binding protein assay (e.g., in vitroplasma binding protein assay), if the compound is assessed in plasmafrom a host species, (e.g., spiked into pooled plasma at a concentrationof about 0.01, about 0.1, about 0.5, about 1, about 5, about 10, about50, about 100, about 500, or about 1000 μg/mL), then the % binding ofthe compound to plasma proteins (PPB) is at least about 1%, at leastabout 2%, at least about 3%, at least about 4%, at least about 5%, atleast about 6%, at least about 7%, at least about 8%, at least about 9%,at least about 10%, at least about 11%, at least about 12%, at leastabout 13%, at least about 14%, at least about 15%, at least about 16%,at least about 17%, at least about 18%, at least about 19%, at leastabout 20%, at least about 21%, at least about 22%, at least about 23%,at least about 24%, at least about 25%, at least about 26%, at leastabout 27%, at least about 28%, at least about 29%, at least about 30%,at least about 31%, at least about 32%, at least about 33%, at leastabout 34%, at least about 35%, at least about 36%, at least about 37%,at least about 38%, at least about 39%, at least about 40%, at leastabout 41%, at least about 42%, at least about 43%, at least about 44%,at least about 45%, at least about 46%, at least about 47%, at leastabout 48%, at least about 49%, at least about 50%, at least about 51%,at least about 52%, at least about 53%, at least about 54%, at leastabout 55%, at least about 56%, at least about 57%, at least about 58%,at least about 59%, at least about 60%, at least about 61%, at leastabout 62%, at least about 63%, at least about 64%, at least about 65%,at least about 66%, at least about 67%, at least about 68%, at leastabout 69%, at least about 70%, at least about 71%, at least about 72%,at least about 73%, at least about 74%, at least about 75%, at leastabout 76%, at least about 77%, at least about 78%, at least about 79%,at least about 80%, at least about 81%, at least about 82%, at leastabout 83%, at least about 84%, at least about 85%, at least about 86%,at least about 87%, at least about 88%, at least about 89%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 95.5%, at leastabout 96%, at least about 96.5%, at least about 97%, at least about97.5%, at least about 98%, at least about 98.5%, at least about 99%, atleast about 99.1%, at least about 99.1%, at least about 99.3%, at leastabout 99.4%, at least about 99.5%, at least about 99.6%, at least about99.7%, at least about 99.8%, at least about 99.9%, at least about99.95%, or at least about 99.99%. The host species can be, for example,mouse (e.g., C57BL6), dog (e.g., beagle), minipig (e.g., Gottingenminipig), sheep, monkey (e.g., Cynomolgus monkey), human, or anysuitable animal disclosed herein.

In some embodiments, in a plasma binding protein assay (e.g., in vitroplasma binding protein assay), if the compound is assessed in plasmafrom a host species, (e.g., spiked into pooled plasma at a concentrationof about 0.01, about 0.1, about 0.5, about 1, about 5, about 10, about50, about 100, about 500, or about 1000 μg/mL), then the % binding ofthe compound to plasma proteins (PPB) is at most about 1%, at most about2%, at most about 3%, at most about 4%, at most about 5%, at most about6%, at most about 7%, at most about 8%, at most about 9%, at most about10%, at most about 11%, at most about 12%, at most about 13%, at mostabout 14%, at most about 15%, at most about 16%, at most about 17%, atmost about 18%, at most about 19%, at most about 20%, at most about 21%,at most about 22%, at most about 23%, at most about 24%, at most about25%, at most about 26%, at most about 27%, at most about 28%, at mostabout 29%, at most about 30%, at most about 31%, at most about 32%, atmost about 33%, at most about 34%, at most about 35%, at most about 36%,at most about 37%, at most about 38%, at most about 39%, at most about40%, at most about 41%, at most about 42%, at most about 43%, at mostabout 44%, at most about 45%, at most about 46%, at most about 47%, atmost about 48%, at most about 49%, at most about 50%, at most about 51%,at most about 52%, at most about 53%, at most about 54%, at most about55%, at most about 56%, at most about 57%, at most about 58%, at mostabout 59%, at most about 60%, at most about 61%, at most about 62%, atmost about 63%, at most about 64%, at most about 65%, at most about 66%,at most about 67%, at most about 68%, at most about 69%, at most about70%, at most about 71%, at most about 72%, at most about 73%, at mostabout 74%, at most about 75%, at most about 76%, at most about 77%, atmost about 78%, at most about 79%, at most about 80%, at most about 81%,at most about 82%, at most about 83%, at most about 84%, at most about85%, at most about 86%, at most about 87%, at most about 88%, at mostabout 89%, at most about 90%, at most about 91%, at most about 92%, atmost about 93%, at most about 94%, at most about 95%, at most about95.5%, at most about 96%, at most about 96.5%, at most about 97%, atmost about 97.5%, at most about 98%, at most about 98.5%, at most about99%, at most about 99.1%, at most about 99.1%, at most about 99.3%, atmost about 99.4%, at most about 99.5%, at most about 99.6%, at mostabout 99.7%, at most about 99.8%, at most about 99.9%, at most about99.95%, or at most about 99.99%. The host species can be, for example,mouse (e.g., C57BL6), dog (e.g., beagle), minipig (e.g., Gottingenminipig), sheep, monkey (e.g., Cynomolgus monkey), human, or anysuitable animal disclosed herein.

In some embodiments, in a plasma binding protein assay (e.g., in vitroplasma binding protein assay), if the compound is assessed in plasmafrom a host species, (e.g., spiked into pooled plasma at a concentrationof about 0.01, about 0.1, about 0.5, about 1, about 5, about 10, about50, about 100, about 500, or about 1000 μg/mL), then the % binding ofthe compound to plasma proteins (PPB) is about 0% to about 10%, about10% to about 20%, about 20% to about 30%, about 30% to about 40%, about40% to about 50%, about 50% to about 60%, about 60% to about 70%, about70% to about 80%, about 80% to about 90%, about 90% to about 100%, about0% to about 20%, about 0% to about 30%, about 0% to about 40%, about 0%to about 50%, about 0% to about 60%, about 0% to about 70%, about 0% toabout 80%, about 0% to about 90%, about 10% to about 30%, about 10% toabout 40%, about 10% to about 50%, about 10% to about 60%, about 10% toabout 70%, about 10% to about 80%, about 10% to about 90%, about 10% toabout 100%, about 20% to about 40%, about 20% to about 50%, about 20% toabout 60%, about 20% to about 70%, about 20% to about 80%, about 20% toabout 90%, about 20% to about 100%, about 30% to about 50%, about 30% toabout 60%, about 30% to about 70%, about 30% to about 80%, about 30% toabout 90%, about 30% to about 100%, about 40% to about 60%, about 40% toabout 70%, about 40% to about 80%, about 40% to about 90%, about 40% toabout 100%, about 50% to about 70%, about 50% to about 80%, about 50% toabout 90%, about 50% to about 100%, about 60% to about 80%, about 60% toabout 90%, about 60% to about 100%, about 70% to about 90%, about 70% toabout 100%, or about 80% to about 100%, about 85% to about 99%, about87% to about 99%, about 88% to about 99%, about 89% to about 99%, about90% to about 99%, about 91% to about 99%, about 92% to about 99%, about93% to about 99%, about 94% to about 99%, about 95% to about 99%, about96% to about 99%, about 97% to about 99%, about 98% to about 99%, about85% to about 98%, about 87% to about 98%, about 88% to about 98%, about89% to about 98%, about 90% to about 98%, about 91% to about 98%, about92% to about 98%, about 93% to about 98%, about 94% to about 98%, about95% to about 98%, about 96% to about 98%, about 97% to about 98%, about85% to about 97%, about 87% to about 97%, about 88% to about 97%, about89% to about 97%, about 90% to about 97%, about 91% to about 97%, about92% to about 97%, about 93% to about 97%, about 94% to about 97%, about95% to about 97%, about 96% to about 97%, about 85% to about 96%, about87% to about 96%, about 88% to about 96%, about 89% to about 96%, about90% to about 96%, about 91% to about 96%, about 92% to about 96%, about93% to about 96%, about 94% to about 96%, about 95% to about 96%, about85% to about 95%, about 87% to about 95%, about 88% to about 95%, about89% to about 95%, about 90% to about 95%, about 91% to about 95%, about92% to about 95%, about 93% to about 95%, or about 94% to about 95%. Thehost species can be, for example, mouse (e.g., C57BL6), dog (e.g.,beagle), minipig (e.g., Gottingen minipig), sheep, monkey (e.g.,Cynomolgus monkey), human, or any suitable animal disclosed herein.

EXAMPLES Example 1. In Vitro Plasma Protein Binding of Compound 1 inMouse, Dog, Minipig, Sheep, Monkey, and Human

This study was designed to determine the in vitro binding of Compound 1to plasma proteins in C57BL6J mouse, Beagle dog, Gottingen minipig,sheep, Cynomolgus monkey, and human. Plasma Protein Binding (PPB) wasassessed in pooled male plasma at nominal Compound 1 concentrations of1, 10, and 50 μg/mL. The assay was performed using the ultrafiltrationtechnique and the concentrations of Compound 1 were determined by LCMS/MS analysis. Warfarin was used as positive control to validate eachultrafiltration run.

Using Busher's classification, Compound 1 binding to plasma proteins wasranked as high-to-very high (PPB>85.0%) at 1 μg/mL and high(85.0%≤PPB≤98.0%) at both 10 and 50 μg/mL. A slight concentrationdependence was observed over the concentration range investigated,suggesting the tendency of PPB to become non-linear with increasingCompound 1 concentration. This trend was more evidently in mouse, dog,minipig, and human.

PPB results of Compound 1 at various concentrations are summarized inTABLE 1.

TABLE 1 Compound 1 Nominal concentration Species (μg/mL) % F % PPB Mouse1 <4.6 ± 0.3^(a ) >95.4 ± 0.3^(a ) 10 8.4 ± 1.8 91.6 ± 1.8 50 10.0^(b)90.0^(b) Dog 1 <4.4 ± 0.3^(a ) >95.6 ± 0.3^(a ) 10  7.0^(b) 93.0^(b) 506.5 ± 1.4 93.5 ± 1.4 Minipig 1 <4.4 ± 0.3^(a ) >95.6 ± 0.3^(a ) 10 5.3 ±0.3 94.7 ± 0.3 50  8.3^(b) 91.7^(b) Sheep 1 <2.8 ± 0.4^(a ) >97.2 ±0.4^(a ) 10 4.2 ± 0.5 95.8 ± 0.5 50 3.8 ± 0.4 96.2 ± 0.4 Monkey 1 <3.8 ±0.5^(a ) >96.2 ± 0.5^(a ) 10 4.7 ± 0.2 95.3 ± 0.2 50  4.8^(b) 95.2^(b)Human 1 <3.1 ± 0.2^(a ) >96.9 ± 0.2^(a ) 10 6.2 ± 0.4 93.8 ± 0.4 50 6.4± 0.5 93.6 ± 0.5 ^(a)Since Compound 1 concentration in the ultrafiltratesample was less than the lower quantification limit (LLOQ = 50 ng/mL),the fraction unbound is calculated as F % < [LLOQ/(C₀ · R)]*100, and PPBexpressed as PPB % >100-F %; C₀ is the Compound 1 initial concentrationand R is the recovery ^(b)SD not calculated since N ≤ 2

Test Systems

Male C57BL6J mouse, male Beagle dog, male Gottingen minipig, male sheep,male Cynomolgus Monkey, and male human plasma was purchased from BIOIVT(UK). Preclinical plasma was pooled from at least 3 different non-fastedanimals, whereas human plasma was obtained from at least 3 healthy andfasted volunteers.

K₃EDTA was used as the anti-coagulant for both the species.

All plasma was stored at 20° C. and thawed only once on the day of theexperiment. No pH correction was applied before the experiment.

Experimental Design

In this study the binding of Compound 1 to mouse, dog, minipig, sheep,monkey, and human plasma proteins was determined in triplicate at thenominal concentrations of 1, 10, and 50 μg/mL using the ultrafiltrationtechnique.

The separation of unbound Compound 1 from plasma proteins was performedusing Centrifree® ultrafiltration devices with a 30,000 NMWL regeneratedcellulose membrane.

The suitability of ultrafiltration as method for protein bindingdetermination of Compound 1 was assessed.

Warfarin was used as a control compound at the single concentration of4.1 μM in male human plasma (n=3) and the control was tested in parallelwith the test item on each ultrafiltration run to confirm the correctexperimental performance.

Methods and Procedures Preparation of Phosphate Buffered Saline (PBS).

Phosphate buffered saline (PBS) was prepared at the concentration of0.146 M. The pH was measured and found to be 7.40. PBS was stored at 4°C.

Compound 1 Spiking Solutions.

The stock solution (SS) containing Compound 1 at 10 mg/mL was preparedon. SS was further diluted in PBS supplemented with 0.2% (v/v) formicacid to obtain the working solution WS1, 100 μg/mL. Compound 1 SS andCompound 1 WS were stored at −80° C.

Preparation of Warfarin Solutions.

A stock solution of warfarin (SS_W) was prepared in water:acetonitrile(50:50, v/v) at a nominal concentration of 3.24 mM (corresponding to 1mg/mL). This solution was further diluted in the same solvent to obtainthe working solution WS_W, 410 μM, which was used for plasma spiking.

Warfarin SS_W was stored at 4° C. (for not more than 21 days, as permethod sheet). Warfarin WS_W was freshly prepared on eachultrafiltration occasion.

Plasma Samples Preparation.

An appropriate volume of single aliquots of mouse, dog, minipig, sheep,monkey, and human plasma was spiked with SS 10 mg/mL or WS 100 μg/mL toobtain the target concentrations of 1, 10, and 50 μg/mL. Each sample wasthen mixed by gentle rocking and rotating for approximately 10-15minutes prior to being transferred to ultrafiltration tubes.

The same procedure was performed for the preparation of warfarin controlsample 4.1 μM (corresponding to 1265 ng/mL) in male human plasma.

Method Suitability for Ultrafiltration.

To assess the suitability of the ultrafiltration technique, experimentsto determine potential non-specific binding to the filter membrane andtest item stability in plasma and PBS over the incubation time wereconducted.

Assessment of Non-Specific Binding of Compound 1 to UltrafiltrationEquipment.

An appropriate amount of PBS was spiked at three test itemconcentrations (1, 10, and 50 μg/mL). The samples were mixed for 1-2 minat room temperature.

Triplicate aliquots of each spiked sample were transferred intoultrafiltration tubes and spun at 1500 g in a Biofuge centrifuge with afixed angle rotor at room temperature, until all loaded volume haspassed through the filter.

Prior to centrifugation (t₀), triplicate aliquots from each sample werecollected and Compound 1 concentrations (C₀) were determined by LC-MS/MSanalysis. After centrifugation, triplicate aliquots of the bottomultrafiltrate (C_(u)) were retained and then analyzed by LC-MS/MS.

Assessment of Compound 1 Stability in Plasma and PBS.

The stability of Compound 1 was assessed at two test item concentrations(1 and 50 μg/mL) in all plasma species and in PBS by incubating eachmatrix for 15 min at 37° C. plus 4 hours at room temperature (time whichcovers incubation, ultrafiltration and sample treatment for analysis).Compound 1 concentrations were determined by LC-MS/MS analysis at botht₀ and t_(4h).

Ultrafiltration Procedure.

The in vitro binding of Compound 1 to plasma proteins was determined atthe three concentrations 1, 10, and 50 μg/mL.

To prevent the non-specific binding of Compound 1 to the ultrafiltrationtubes, the inner wall was rinsed with 10% (v/v) Triton X-100 solutionand left to dry for at least 1 hour before use.

Triplicate aliquots of each spiked plasma were transferred intoultrafiltration tubes. The ultrafiltration tubes were spun at roomtemperature for 20 min at 1500 g in a Biofuge centrifuge with a fixedangle rotor.

Prior to centrifugation, triplicate aliquots of spiked plasma wereretained to give the concentration of Compound 1 in plasma (C₀)post-ultracentrifugation. After centrifugation, an appropriate aliquotof the top retentate (C_(r)) and bottom ultrafiltrate (C_(u)) was thenremoved for LC-MS/MS analysis.

The whole ultrafiltration devices, the ultrafiltrate reservoirs and thefiltrate cups were weighed before adding the sample, after adding thesample (only for the whole ultrafiltration devices) and at the end ofthe centrifugation.

The same procedure was performed for warfarin control samples.

Determination of Protein Content in the Ultrafiltration Samples.

After collection, each ultrafiltration sample was checked for proteincontent to assess a possible protein contamination and then to confirmthe filter integrity over ultrafiltration. The presence of proteins wasassessed in the ultrafiltrate using the Bradford method.

A calibration standard curve of bovine serum albumin was prepared at thefollowing concentrations: 0, 50, 100, 200, 400, 600, 800, and 1000μg/mL.

Calibration standard samples and ultrafiltrate samples were dispensed ina 96-well plate (6 μL/well) and supplemented with Coomassie BrilliantBlue G Solution (300 μL). After 1 min at room temperature, theabsorbance of each well was measured at λ=595 nm by an absorbancemicroplate reader

Sample Treatment and Analysis.

Appropriate aliquots of spiked plasma (collected prior to and afterultrafiltration) and ultrafiltration samples (collected afterultrafiltration) were supplemented with an equal volume (matrixmatching) of drug-free PBS or drug-free plasma, respectively.

After matrix matching, plasma and ultrafiltration samples were extractedand analysed by LC-MS/MS.

Storage of Samples.

All samples were collected into uniquely identifiable containers,labelled with the study number, sample type, nominal sampleconcentration and incubation time. Compound 1 samples and Warfarinsamples were stored at −80° C.

Data Handling and Analysis Plasma Protein Binding CalculationsNon-Specific Binding (NSB)

NSB to ultrafiltration tubes was determined from the Compound 1concentrations in PBS samples prior to centrifugation (C₀) and in thecollected ultrafiltration samples after centrifugation (C_(u)).

${{NSB}\mspace{14mu}\%} = {\frac{C_{0} - C_{u}}{C_{0}} \cdot 100}$

where:

C₀=concentration of Compound 1 before the ultrafiltration, ng/mL

C_(u)=concentration of Compound 1 after ultrafiltration, ng/mL

Stability Assessment.

The extent of any potential loss of Compound 1 after incubation inplasma (or PBS) at room temperature as long as the duration of theexperiment (t_(4h)) was calculated as follows:

${\%\mspace{14mu}{difference}\mspace{14mu}\left( {t_{4h} - t_{0}} \right)} = {\frac{C_{4h} - C_{0}}{C_{0}} \cdot 100}$

Where:

C₀=concentration in matrix (plasma or PBS) at to, ng/mL

C_(4h)=concentration in matrix (plasma or PBS) after t_(4h), ng/mL

Plasma Protein Binding.

The % binding of Compound 1 (B % or PPB %) to plasma proteins wasdetermined using the following equations:

B  % = 100 − (F  %) $F = \frac{C_{u}}{R \cdot C_{0}}$

Where:

F=free fraction

R=recovery

The same calculations were performed for warfarin control sample.

Total Recovery.

Comparing the concentration of Compound 1 in the ultrafiltrate and inthe retentate samples (after ultrafiltration) with that of Compound 1 inthe samples before the ultrafiltration, a recovery value was obtained,as described in the following formula:

${R\mspace{14mu}\%} = {\left( \frac{{C_{u} \times V_{u}} + {C_{r} \times V_{r}}}{C_{0} \times V_{0}} \right) \cdot 100}$

Where:

V₀=initial volume of plasma sample, before ultrafiltration (mL)V_(r)=volume of retentate, after ultrafiltration (mL)V_(u)=volume of ultrafiltrate, after ultrafiltration (mL)C₀=concentration in to plasma sample (ng/mL)C_(r)=concentration in the retentate sample (ng/mL)C_(u)=concentration in the ultrafiltrate sample (ng/mL)The density of each matrix (plasma, retentate and ultrafiltrationsample) was assumed 1.The same calculations were performed for warfarin control sample.

Protein Binding Acceptance Criteria.

NSB≤20% is acceptable. Compound 1 was considered stable in plasma andPBS if the percentage difference between concentration after t_(4h) andto is within ±15%. Protein binding of the positive control warfarinshould be ≥98.0%. The protein concentration measured afterultrafiltration should be ≤0.3 mg/mL.

Recovery (R) values (Range of Recovery, %) were assessed as detailed asfollows: R≥200 or R≤50 means Experimental data not valid; 50≤R<80 or120<R≤200 means Experimental data potentially unreliable; and 80≤R≤120means Experimental data valid.

Results Non-Specific Binding (NSB) Assessment.

On average, NSB to filter membrane represented 20.5%, 6.2%, and 2.2% at1, 10, and 50 μg/mL Compound 1 concentrations, respectively, indicatingan increase of NSB contribution with decreasing concentration (which isthe typical trend for compounds showing NSB). Since the NSB was notgreater than around 20% (in the worst-case scenario), theultrafiltration technique was deemed fit for purpose.

The results of NSB assessment are presented in TABLE 2.

TABLE 2 Concentration of Compound 1 C₀ C₀ Nominal Actual ConcentrationConcentration C_(u) Average (ng/mL) (ng/mL) (ng/mL) % NSB  1000  950 761 20.5%  886  760  861  626 Mean ± SD 899 ± 46  716 ± 78 10000 9978 9450  6.2% 9479  9550 10442   8980 Mean ± SD 9966 ± 482 9327 ± 30550000 49368  48269  2.2% 49917  50900 51746  48414 Mean ± SD 50344 ±1245 49195 ± 1479

Determination of Compound 1 Stability in Plasma and PBS.

Compound 1 was stable (i.e., with a percentage loss not greater than15%) in mouse, dog, sheep, monkey, and human plasma as well as PBS whenincubated at room temperature for up to 4 hours at both 1 and 50 μg/mL.

Compound 1 was stable in minipig plasma at 1 μg/mL, whereas a slightinstability (characterized by a percentage loss of 22.5%) was observedat 50 μg/mL.

The results of Compound 1 stability in plasma and in PBS samples arepresented in TABLE 3.

TABLE 3 C₀ Actual Concentration Nominal Concentration (ng/mL) Matrix(ng/mL) C₀ C_(4 h) Mouse 1000 1136 1288 1036 1422 1283 1266 Mean ± SD1151 ± 124 1325 ± 84  % Difference (4 h − 0 h) 15.1% 50000 80435  66779 67255  62362  70127  63006  Mean ± SD 72606 ± 6931 64049 ± 2386 %Difference (4 h − 0 h) −11.8%  Dog 1000  962 1054 1033 1126 1156  870Mean ± SD 1050 ± 98  1017 ± 132 % Difference (4 h − 0 h) −3.2% 5000042457  39519  45231  48050  44416  38581  Mean ± SD 44035 ± 1426 42050 ±5217 % Difference (4 h − 0 h) −4.5% Minipig 1000  981 1021  997 12151141 1040 Mean ± SD 1039 ± 88 1092 ± 107 % Difference (4 h − 0 h)  5.1%50000 67014  54551  70687  59236  71484  48346  Mean ± SD 69728 ± 238454044 ± 5463 % Difference (4 h − 0 h) −22.5%  Sheep 1000 1199 1425 16221550 1664 1692 Mean ± SD 1495 ± 257 1556 ± 133 % Difference (4 h − 0 h) 4.1% 50000 74884  85924  73441  89388  84447  83543  Mean ± SD 77590 ±5981 86285 ± 2939 % Difference (4 h − 0 h) 11.2% Monkey 1000 1075 11081240 1188 1192 1160 Mean ± SD 1169 ± 85  1152 ± 41  % Difference (4 h −0 h) −1.5% 50000 43353  46034  49384  47590  42860  49370  Mean ± SD45199 ± 3633 47664 ± 1670 % Difference (4 h − 0 h)  5.5% Human 1000 10091082 1005 1123 1035 1071 Mean ± SD 1016 ± 17  1092 ± 27  % Difference (4h − 0 h)  7.5% 50000 51417  50163  53571  59910  54627  48923  Mean ± SD53205 ± 1636 52999 ± 6017 % Difference (4 h − 0 h) −0.4% PBS 1000 833769 824 676 734 600 Mean ± SD 797 ± 55 682 ± 85 % Difference (4 h − 0 h)−14.5%  50000 49419  51395  50911  49815  50858  46452  Mean ± SD 50396± 846 49221 ± 2525 % Difference (4 h − 0 h) −2.3%

Determination of Compound 1 Plasma Protein Binding.

The extent of PPB has been defined according to Busher's classification:very high (PPB>98.0%), high (85.0%≤PPB≤98.0%), and medium-to-low(PPB<85.0%).

PPB results of Compound 1 at various concentrations (1, 10, and 50μg/mL) are summarized in TABLE 1 and fully reported in TABLE 4.

TABLE 4 Concentration of Compound 1 Nominal Experi- C₀ Actual C₀ C_(r)C_(u) ment Species (ng/mL) (ng/mL) (ng/mL) (ng/mL) F % PPB % R % 1Minipig 1000 1047 1441 NQ <4.4^(a) >95.6^(a) >107.8 1010 1333 NQ<4.7^(a) >95.3^(a) >104.3 1164 1539 NQ <4.1^(a) >95.9^(a) >103.7 Mean ±1074 ± 80  1437 ± 103 NA <4.4 ± 0.3^(a) >95.6 ± 0.3^(a) >105.3 ± 2.2  SD10000 9553 12216  482 5.0 95.0 101.1  9147 11904  501 5.3 94.7 102.5 11182  13494  599 5.7 94.3 94.7 Mean ± 9961 ± 1077 12538 ± 843  527 ± 635.3 ± 0.3 94.7 ± 0.3 99.5 ± 4.2 SD 50000 46736  51751  2946  7.2 92.887.0 54997  63661  NR NC NC NC 48959  48394  3641 9.4 90.6 78.8 Mean ±50231 ± 4275 54602 ± 8023 3293^(b )  8.3^(b)  91.7^(b)  82.9^(b) SD 2Dog 1000 1153 1611 NQ <4.2^(a) >95.8^(a) >103.5 1187 1390 NQ <4.8^(a)>95.2^(a)  >88.6 1248 1580 NQ <4.3^(a) >95.7^(a)  >92.5 Mean ± 1196 ± 481527 ± 120 NA <4.4 ± 0.3^(a) >95.6 ± 0.3^(a) >94.9 ± 7.7 SD 10000 871610656  NR NC NC NC 8039 10701  577 7.0 93.0 102.3   4234^(#) 11215  NRNC NC NC Mean ±  8378^(b) 10857 ± 311  577^(b)  7.0^(b)  93.0^(b)102.3^(b) SD 50000 42816  50194  2400  6.5 93.5 86.2 38278  48731  2823 7.9 92.1 93.5 40637  52325  1931  5.1 94.9 93.2 Mean ± 40577 ± 226950416 ± 1807 2385 ± 446 6.5 ± 1.4 93.5 ± 1.4 91.0 ± 4.1 SD 3 Human 10001480 2074 NQ <3.1^(a) >96.9^(a) >109.0   1670 2024 NQ <3.2^(a)>96.8^(a) >92.5  1466 2216 NQ <2.9^(a) >97.1^(a) >116.4   Mean ± 1539 ±114 2105 ± 100 NA <3.1 ± 0.2^(a) >96.9 ± 0.2^(a) >106.0 ± 12.2 SD 1000010965  11121  573 6.6 93.4 78.8 11464  14447  663 5.9 94.1 97.6 11833 13710  654 6.2 93.8 89.7 Mean ± 11421 ± 436  13093 ± 1747 630 ± 50 6.2 ±0.4 93.8 ± 0.4  88.7 ± 9.5 SD 50000 49892  59456  3060  6.5 93.5 93.754867  60928  2859  5.9 94.1 88.1 53454  56932  3102  6.9 93.1 84.5 Mean± 52738 ± 2564 59105 ± 2021 3007 ± 130 6.4 ± 0.5 93.6 ± 0.5  88.7 ± 4.7SD 4 Monkey 1000 1577 2010 NQ <3.3^(a) >96.7^(a) >96.8  1482 1760 NQ<3.8^(a) >96.2^(a) >89.3  1603 1533 NQ <4.3^(a) >95.7^(a) >72.4  Mean ±1554 ± 64  1768 ± 239 NA <3.8 ± 0.5^(a) >96.2 ± 0.5^(a)  >86.1 ± 12.5 SD10000 7655 9163 339 4.9 95.1 90.7 8718 10679  384 4.8 95.2 92.5 841910083  343 4.4 95.6 91.5 Mean ± 8264 ± 548 9975 ± 764 355 ± 25 4.7 ± 0.295.3 ± 0.2  91.6 ± 0.9 SD 50000 44699  39892^(# ) 1994  NR NR NR 42296 54604  2033  4.9 95.1 98.9 39204  51902  1844  4.7 95.3 99.9 Mean ±42066 ± 2755 53253^(b)  1957 ± 100  4.8^(b)  95.2^(b)  99.4^(b) SD 5Mouse 1000 1569 1569 NQ <4.4^(a) >95.6^(a) >72.1  1378 1560 NQ <4.5^(a)>95.5^(a) >81.0  1524 1455 NQ <5.0^(a) >95.0^(a) >66.2  Mean ± 1490 ±100 1528 ± 63 NA <4.6 ± 0.3^(a) >95.4 ± 0.3^(a)  >73.1 ± 7.5 SD 1000011029  12959  988 10.4  89.6 86.3 10253  13494  747 7.7 92.3 94.8 12758 15591  782 7.1 92.9 86.4 Mean ± 11346 ± 1282 14015 ± 1391 839 ± 130 8.4± 1.8 91.6 ± 1.8  89.2 ± 4.9 SD 50000 57461  64540  4624  9.7 90.3 82.860430  125704^(#  ) 4164  NR NR NR 58087  63492  4779  10.2  89.8 80.7Mean ± 58659 ± 1565 64016^(b ) 4522 ± 320 10.0^(b)  90.0^(b)  81.8^(b)SD 6 Sheep 1000 2221 2024 NQ <3.2^(a) >96.8^(a) >69.7  2027 2585 NQ<2.6^(a) >97.4^(a) >96.1  1973 2483 NQ <2.7^(a) >97.3^(a) >94.8  Mean ±2074 ± 131 2364 ± 299 NA <2.8 ± 0.4^(a) >97.2 ± 0.4^(a)  >86.9 ± 14.9 SD10000 16378  16595  610 4.8 95.2 77.5 17745  20050  602 3.9 96.1 86.317360  18129  528 3.8 96.2 79.1 Mean ± 17161 ± 705  18258 ± 1731 580 ±45 4.2 ± 0.5 95.8 ± 0.5  81.0 ± 4.7 SD 50000 81340  92345  2417  3.596.5 86.1 77645  99298  2896  3.9 96.1 96.5 85273  97221  3108  4.2 95.887.0 Mean ± 81419 ± 3814 96288 ± 3569 2807 ± 354 3.8 ± 0.4 96.2 ± 0.4 89.9 ± 5.8 SD NQ = not quantifiable, NA = not applicable, NR = notreportable, NC = not calculable ^(#)= Outlier, possibly due to asampling error (value excluded from the mean calculation) ^(a)SinceCompound 1 concentration in the ultrafiltrate samples was less than thelower quantification limit (LLOQ = 50 ng/mL), the fraction unbound iscalculated as F % < [LLOQ/(C₀ · R)]*100, and PPB expressed as PPB % >100-F %; ^(b)SD not calculated since N ≤ 2

A slight concentration dependence was observed over the concentrationrange investigated. The observation suggested the tendency of PPB tobecome non-linear with increasing Compound 1 concentration, moreevidently in mouse, dog, minipig and human.

Average PPB of warfarin was ≥99.2% for all the experiments, thusvalidating each ultrafiltration run (see TABLE 5).

TABLE 5 Concentration of Warfarin Nominal Actual C₀ C₀ c_(r) C_(u)Experiment (ng/mL) (ng/mL) (ng/mL) (ng/mL) F % PPB % R % 1 1265 11651302 7.52 0.7 99.3 86.4 (PPB in 1168 1356 6.82 0.6 99.4 90.2 Minipig)1181 1517 6.73 0.6 99.4 100.0  Mean ± 1171 ± 8.4 1392 ± 112 7.02 ± 0.40.7 ± 0.09 99.3 ± 0.09 92.2 ± 7.0 SD 2 1265 1187 1265 6.71 0.7 99.3 83.6(PPB in 1139 1429 6.24 0.6 99.4 98.8 Dog) 1139 1412 6.06 0.5 99.5 97.2Mean ± 1155 ± 28 1369 ± 90 6.34 ± 0.3 0.6 ± 0.07 99.4 ± 0.07 93.2 ± 8.4SD 3 1265 1115 1274 8.24 0.8 99.2 88.0 (PPB in 1145 1350 8.35 0.8 99.289.6 Human) 1141 1364 8.57 0.8 99.2 91.9 Mean ± 1134 ± 16 1330 ± 49 8.39± 0.2 0.8 ± 0.01 99.2 ± 0.01 89.8 ± 1.9 SD 4 1265 1191 1422 7.44 0.799.3 94.0 (PPB in 1155 1401 6.58 0.6 99.4 94.8 Monkey) 1219 1289 6.490.6 99.4 85.2 Mean ± 1188 ± 32 1371 ± 72 6.84 ± 0.5 0.7 ± 0.03 99.4 ±0.0 91.4 ± 5.3 SD 5 1265 1178 1429 8.88 0.8 99.2 93.6 (PPB in 1159 14906.81 0.6 99.4 101.6  Mouse) 1165 1431 7.08 0.6 99.4 95.5 Mean ± 1167 ±10 1450 ± 35 7.59 ± 1.1 0.7 ± 0.1 99.3 ± 0.1 96.8 ± 4.3 SD 6 1265 11931478 8.12 0.7 99.3 96.8 (PPB in 1160 1389  5.89^(a) NC NC NC Sheep) 11931412 6.87 0.6 99.4 92.8 Mean ± 1182 ± 19 1426 ± 46 6.96 ± 1.1  0.7^(b) 99.3^(b)  94.8^(b) SD ^(a)Sample protein contaminated (proteincontent >300 μg/mL). The corresponding % F, % PPB and % R are notcalculated (NC) and excluded from the mean ^(b)SD not calculated since N≤ 2

No protein contamination was detected in any ultrafiltration Compound 1samples.

Bioanalytical Results.

The matrix-matched samples were analyzed using three qualifiedbioanalytical LC-MS/MS methods: one for warfarin and two for Compound 1(one for minipig and one for human, cynomolgus monkey, dog, sheep, andmouse).

Carry over was assessed throughout the study phase by injection of oneor more blank samples after a high concentration standard. Carry overwas more than 25% of the response at the LLOQ for Compound 1 and lessthan 5% for the internal standard. Carry over was more than 25% of theresponse at the LLOQ for warfarin and less than 5% for the internalstandard. Study samples were analyzed following the expectedconcentration to avoid potential impact on the final results.

Compound 1 and warfarin concentrations in study samples were determinedfrom the appropriate calibration plots within BioLims.

Samples with unexpected results were re-assayed in duplicate. In somecases, results were not reportable (NR).

Conclusions

In agreement with Busher's classification, Compound 1 binding to plasmaproteins was ranked as high-to-very high (PPB>85.0%) at 1 μg/mL and high(85.0%≤PPB≤98.0%) at both 10 and 50 μg/mL. A slight concentrationdependence was observed over the concentration range investigated,suggesting the tendency of PPB to become non-linear with increasingCompound 1 concentration. This trend was more evident in mouse, dog,minipig, and human.

Example 2. Tissue Distribution of Total Radioactivity in the CynomolgusMonkey Following Single Intravenous Bolus Administration of Compound 1

The objective of this study was to assess tissue distribution,disposition, and pharmacokinetics (PK) of [¹⁴C]-Compound 1 drug relatedmaterial following a single intravenous (IV) administration to the malecynomolgus (Macaca fascicularis) monkeys at the target dose level of 5mg/kg using quantitative whole-body autoradiography (QWBA). Thecynomolgus monkey is representative of the distribution and excretion ina human. Three animals were placed into metabolism cages after dosingand kept up to 4, 12, and 168 h post dose, respectively.

Disposition of [¹⁴C]-Compound 1 was investigated in one animal only (No.103, 168h). Total radioactivity was measured in urine, feces, and cagerinse.

Pharmacokinetic profiles were obtained in blood and plasma collectedfrom each animal at selected timepoints post-dose up to the terminaltimepoint.

Following euthanasia at 4, 12, and 168 h post dose, tissue distributionof [¹⁴C]-Compound 1 was investigated using quantitative whole bodyautoradiography.

Following IV bolus administration of [¹⁴C]-Compound 1, very limitedamounts of administered radioactivity (4.2% of the dose) were excretedover 168 h post-dose. This observation indicated that most of the dosewas retained in the tissues: 1.5% and 1.7% of the dose was recovered inurine and feces, respectively. Cage rinse, which relates to urinaryexcretion, accounted for another 0.9% of the dose.

Total radioactivity in blood and plasma was quantifiable up to terminaltimepoint in all animals. Over 168 h post dose (animal 103), systemicexposure to total radioactivity, measured as area under the plasmaconcentration time curve (AUC) from the start of dosing to the lastquantifiable time point (T_(last)) (AUC_(0-t)), was 231000 and 205000h·ng-eq/g in blood and plasma, respectively, while maximum concentration(C_(max)) was 7510 ng-eq/g (C₀ 9260 ng-eq/g) and 11500 ng-eq/g (C₀ 14000ng-eq/g), respectively. Total radioactivity showed higher partitioninginto plasma than blood (blood to plasma ratio 0.6-0.8) except for 168 hwhere a ratio of 2.1 was observed.

Clearance (CL) was 6.69 mL/h/kg (blood) and 14.0 mL/h/kg (plasma) andwas significantly lower than were hepatic and renal blood flow rates(which are approximately 2616 and 1656 mL/h/kg). Consistently, meanvolume of distribution at steady state (V_(ss)), accounting for 2480mL/kg (blood) and 2780 mL/kg (plasma), was greater than total body waterin cynomolgus monkey (which is approximately 693 mL/kg), indicating amoderate (0.6 to 5 L/kg) volume of distribution.

In general, [¹⁴C]-Compound 1 drug-related material was widelydistributed throughout the whole body and quantifiable in all tissues upto the last timepoint sampled. The distribution pattern in tissues wascomparable between selected timepoints over 168 h post-dose. Very highconcentrations were observed in kidney cortex, joints (most likelyconnected to cartilage), and liver, suggesting potential accumulation inthese tissues.

Other tissues that showed notable uptake were: salivary glands, bonesurface, pancreas, hair follicles, large intestine mucosa, aortic wall,small intestine mucosa, adrenal gland, stomach mucosa, spleen, bonemarrow, lymph nodes, and thymus.

Moderate brain penetration was observed (brain to blood ratio<1): totalradioactivity distributed quite uniformly in relevant sub-regions(cerebellum, olfactory bulb, thalamus, caudate putamen, cerebral cortex,and substantia nigra), but the highest concentrations were measured inthe lateral ventricle, most likely in the choroid plexus.

Finally, radioactivity was also measured in tissues containing melanin(e.g., hair follicles and, most importantly, the uveal tract in the eye)indicating potential binding to melanin.

Radiolabeled Test Item.

[¹⁴C]-Compound 1 (radiolabel was given by conjugation to double¹⁴C-labelled glycine)

Specific activity: 29.3 μCi/mg (1.08 MBq/mg)

Radiochemical Purity: 73.7%

The radiolabeled test item, supplied as powder (88.8 MBq correspondingto 81.9 mg), was dissolved in the vehicle (8.9 mL) to obtain aradioactive stock solution (SS) at a nominal concentration of 10 MBq/mL(9.2 mg/mL). Actual radioactivity concentration and radiochemical purityof SS was determined prior to use by liquid scintillation counting(LSC). SS was stored at −80° C.

Vehicle (Control).

DPBS: Dulbecco's Phosphate Buffered Saline (modified, without calciumchloride and magnesium chloride, liquid, sterile-filtered).

pH 7.1-7.5.

Osmolality: 275-304 mOs/kg.

Storage Conditions: Below 30° C. (Ambient temperature, AT), 2-8° C. whenopened.

Test Item Formulation.

Target dose level: 5.0 mg/kg

Target radioactive dose: 147 μCi/kg (5.42 MBq/kg)

Dose volume: 2.0 mL/kg

Nominal concentration: 2.5 mg/mL

Nominal radioactivity concentration: 73.3 μCi/mL (2.71 MBq/mL)

Method: The final formulation was prepared the day beforeadministration. SS was thawed at ambient temperature, heated atapproximately 65° C. for 5 minutes and then allowed to cool down to roomtemperature under magnetic stirring. An appropriate aliquot of SS wasdiluted in the required volume of vehicle and kept under magneticstirring at room temperature overnight. At the stated specific activity,no additional isotopic dilution with non-radiolabeled test item wasrequired.

Stability of the final formulation: The heating step was assessed inadvance to confirm that [¹⁴C]-Compound 1 stability was not affected. Thestability at ambient temperature was assessed over 24 hours in order toallow for formulation preparation on the day before administration.

Storage conditions of the final formulation: Ambient temperature up to24 hours, prior to dosing.

Residual Test Item Formulation: The residual formulation was stored at−20° C.

Test System Characterization of Test System.

Number on Study: 3 males

Age: Between 2 and 2.5 years

Body Weight: 2.40 kg, 2.24 kg, 2.29 kg

Minimum Acclimatization: At least 21 days (prior to Day 1)

Monkey was used as one of the non-rodent species required in toxicologystudies by test guidelines. The cynomolgus monkey was chosen because thenon-human primate blood brain barrier is more similar to that of a humanthan to a dog blood brain barrier.

Temperature and humidity were recorded daily during the study phases.Actual mean measurements (with ranges) were 21.3° C. and 52.7%. Averagetemperature and relative humidity were always within the acceptableranges during the in life phase: 21-23° C. (20-24° C. for less than 24hours acceptable) and 45-65% (40-70% for less than 24 hours acceptable),respectively.

Diet, Water, and Environmental Enrichment.

Diet was offered ad libitum throughout the study except forapproximately 1 hour before and after dosing. A fruit, vegetable, andforaging mix supplement was also given.

Water, filtered from normal domestic water, was offered ad libitumthroughout the study.

Monkeys had access to specific environmental enrichment devices in eachcage. Social interaction between animal and staff was ensured for anadequate time twice a day on working days, and once a day over theweekend.

Experimental Design

Three naïve male Cynomolgus monkeys each received a single IV bolusadministration of [¹⁴C]-Compound 1 at a target dose of 5 mg/kg.Following dose administration, animals were placed into metabolic cagesand the following matrices were collected as outlined in TABLE 6.

TABLE 6 Animal Animal No./Sex Matrix No Time-point (h) 3/males Blood 1015 min, 15 min, 30 min, 1 h, 2 h, 4 h 102 5 min, 15 min, 30 min, 1 h, 2h, 3 h, 4 h, 6 h, 8 h 103 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6h, 8 h, 12 h, 24 h, 168 h Tissues 101  4 h post-dose (terminaltimepoint) (carcass) 102  12 h post-dose (terminal timepoint) 103 168 hpost-dose (terminal timepoint) Urine 101/102 Samples not available atterminal timepoint 103 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 hFeces 101/102 Samples not available at terminal timepoint 103 24 h, 48h, 72 h, 96 h, 120 h, 144 h, 168 h

Levels of total radioactivity were determined by liquid scintillationcounting (LSC) in the following matrices: blood, plasma, urine, feces,and cage rinse.

At the terminal timepoint (4 h, 12 h, and 168 h post-dose,respectively), animals were euthanized, and the tissue distribution oftotal radioactivity was evaluated using QWBA.

Intra-organ distribution of total radioactivity was evaluated in brain(cerebellum, olfactory bulb, thalamus, caudate putamen, cerebral cortex,and substantia nigra), eye and kidney. Sections were collected using acryomacrotome, freeze-dried, and exposed to imaging plates. Theresulting electronic whole-body autoradiograms were evaluated for thequantitative assessment of radioactivity in the tissues.

Radioactivity data in blood, plasma and tissues were subject tonon-compartmental analysis for evaluation of appropriate pharmacokineticparameters, where feasible.

Methods and Procedures Assessment of Test Item Stability.

The stability of the test item was assessed, by measuring theradiochemical purity, under the following conditions prior to initiatingin-vivo experimental activities.

An aliquot of the SS was heated at approximately 65° C. for 5 minutes toconfirm that this step, during formulation preparation, would not affect¹⁴C label stability.

The stability of the final formulation was assessed at room temperaturefor 4 and 24 hours.

A preliminary assessment of [¹⁴C]-Compound 1 stability in monkey bloodwas also performed: a volume of monkey blank blood was spiked with anappropriate aliquot of radiolabeled SS and incubated at 37° C. for 4 and24 hours.

As radioactivity could not be extracted using a standard approach,measurement of radiochemical purity after incubation in blood andassessment of stability in biological matrices were not possible.

Characterization of Stock Solution and Formulation.

The radioactivity concentration of the stock solution and doseformulation was determined as follows: three weighed aliquots weredispensed into glass vials, appropriately diluted with DPBS. Threeweighed aliquots were then removed from each dilution, supplemented withliquid scintillant and radioassayed by LSC in order to determine theactual radioactive concentration and homogeneity of the solutions.

Dose Administration.

The dose formulation was administered intravenously, as a bolus, throughthe caudal vein.

Individual dose volumes were adjusted based on body weight of theanimals on the day of dose administration. Doses were dispensed intopre-weighed syringes, which were weighed prior to and following doseadministration. The actual dose received by each animal was calculatedfrom dose concentration, weight of dose administered and animal weight.

Sample Collection. Phase 1: Excretion Balance.

Following administration, urine and feces were collected quantitativelyat selected times post-dose and kept refrigerated, over wet ice, duringcollection.

On a daily basis and/or at the end of the collection period, metaboliccages were rinsed with ethanol/water (50:50, v/v) and the wash retainedfor quantitative determination of radioactivity.

Cage debris were collected and pooled by animal over the entire periodof collection.

Phase 2: Pharmacokinetics.

Following administration, systemic blood was removed from the cephalicvein, at each selected time point post-dose and transferred into tubescontaining K₃EDTA as anti-coagulant (actual times of bleeding wererecorded). Following collection, all blood samples were thoroughly mixedand placed on wet ice. Plasma samples were prepared within two hours ofblood collection.

Liquid Scintillation Counting Analysis.

Triplicate aliquots of liquid samples (e.g., dose formulations, urine,plasma and cage wash) were counted directly in liquid scintillant.

Feces were homogenized in a suitable quantity of ultrapure water(approximate ratio 1:2, w/v) using Stomacher homogenizer. Weighedquadruplicate aliquots of homogenates (0.2-0.4 g) were solubilized byaddition of 1 mL of Solvable tissue solubilizer, supplemented twice with200 of 30% hydrogen peroxide (H₂O₂) and incubated at approximately 50°C., until appropriate discoloration was achieved, prior to addition ofscintillant.

Weighed triplicate aliquots of blood (100 μL) were treated as fecalhomogenates.

The remaining blood was centrifuged at 2000 g for 10 min atapproximately 4° C. within two hours of collection and the plasmadecanted into plastic tubes. Aliquots of plasma were radio assayed byLSC. Blood pellets were discarded.

Radioactivity was determined in a Tricarb Series liquid scintillationanalyzer. Quench correction was achieved during sample counting throughthe automatic assignment of a quench indicator (tSIE/AEC) value to thesample. This value was used to interpolate sample counting efficiencyfrom an instrument stored quench curve generated from a series of sealedquenched standards. The interpolated efficiency value was used toautomatically correct LSC data counts per minute (cpm) and obtaindisintegrations per minute (dpm). A suitable scintillation fluid (UltimaGold) was added to each sample prior to radio assaying by LSC for anappropriate time. Background counts were subtracted from quenchcorrected sample counts. According to the background value and counttime, the limit of quantification was calculated.

Tissue Distribution.

At the last timepoint (4 h, 12 h, and 168 h post-dose, respectively)animals were euthanized and the tissue distribution of totalradioactivity was evaluated using quantitative whole-bodyautoradiography.

Sample Collection and Embedding.

Following euthanasia, animals were frozen by immersion into a freezingmixture of hexane/solid CO₂. Animals were left in the freezing mixturefor at least 2 hours, until completely frozen.

Frozen carcasses were embedded, left lateral side uppermost, in a blockof sodium carboxymethylcellulose (approximately 2%, w/v in water).

Sectioning.

Each block was mounted in a CM3600 cryomacrotome (Leica Microsystems)maintained at approximately −20° C. After initial trimming of the block,sagittal whole body sections (40 μm) were obtained at various levelsthrough the carcass. Sections of interest mounted on pressure sensitivetape were left to dehydrate in the cryomacrotome chamber atapproximately −20° C. for approximately 60 h.

Autoradiography.

Freeze dried sections were placed against an image plate (IP), suitablefor ¹⁴C, as long as 4 days. During the exposure period the IPs wereplaced in a copper-lead shielding box for minimizing external backgroundsignals. Following exposure, the IPs were scanned by a laser beam, usinga Fuji FLA5000 phosphor imager system, and the latent image captured andstored on an electronic data file.

For each set of whole body images obtained, the system was calibratedwith biological standards of known radioactivity.

Selected tissues were identified on the image and integrated, using AIDAsoftware, to give a value for tissue concentration. Triplicatemeasurements were made across each selected organ/tissue, when possible,either by multiple integration on a single section or by multipleintegration from a number of different sections.

Results generated from the integration were expressed as Bq/g andsubsequently converted to ng-equivalents of Compound 1/g tissue(ng-eq/g), using dose formulation specific activity.

The limit of quantification (LOQ) for tissue distribution was set as 3times the mean background levels of radioactivity (mean of sevenmeasurements per IP).

For the purposes of quantification, the assumption was that all tissuesanalyzed had a similar density and quench characteristics to blood (usedas calibration standards). Exceptions to this rule were bone and whitefat, where correction factors of 0.48 and 0.84 were applied,respectively, to compensate for quenching of radioactivity.

Calibration Curve.

A block of frozen CMC (approximately 2%, w/v, in water) containingstandards of known radioactivity was prepared on a separate occasion. Aseries of paper straws were fixed in the block and monkey bloodcontaining increasing amounts of a [¹⁴C]-Compound 1 was dispensed intothe straws and frozen. The range of calibration standards was chosen tocover the extent of likely tissue concentration values (actual range56.8-32,274 Bq/g).

Calibration lines were generated for each set of sections exposed on asingle IP by selecting an area of each standard from the scanned imageand assigning the corresponding radioactivity concentration previouslydetermined by LSC.

Results Stock Solution Analysis.

The radioactivity concentration and the radiochemical purity of thestock solution was checked prior to use. Radioactivity concentration(MBq/mL) was 9.52. Radiochemical purity was 93.2%.

Assessment of Test Item Stability.

Compound 1 was stable under the experimental conditions assessed.

Formulation Analysis.

Compound 1 Concentration: 2.33 mg/g (mg of Compound 1+[¹⁴C]-Compound 1)

Radioactivity Concentration: 2.52 MBq/g

Formulation Specific Activity: 1.08 MBq/mg

CV: 1.9% (overall pre- and post-dose)

Difference Pre vs Post dose: 1.6%

Radiochemical Purity: 91.8% (mean pre- and post-dose)

The formulations resulted to be homogeneous prior to and across dosing(CV<5%).

The difference between pre dose and post dose concentration was within±10%.

Administered Dose.

The actual doses of [¹⁴C]-Compound 1 administered to male cynomolgusmonkeys as single IV bolus are presented in TABLE 7.

TABLE 7 Animal Body Weight Formulation Chemical Dose^(a) RadioactiveDose^(b) Number (kg) Administered (g) (mg/Animal) (mg/kg) (MBq/Animal)(MBq/kg) 101 2.29 4.69 10.9 4.77 11.8 5.15 102 2.40 4.83 11.3 4.69 12.25.06 103 2.24 4.51 10.5 4.69 11.3 5.07 Mean 2.31 4.68 10.9 4.72 11.85.09 ±SD 0.08 0.16  0.4  0.047  0.4  0.051 ^(a)Chemical doseconcentration = 2.33 mg [¹⁴C]-Compound 1 + Compound 1/g of formulation^(b)Radioactive dose concentration = 2.52 MBq/g of formulation

Mean Actual Dose: 4.72±0.05 mg of total Compound 1/kg (mg of Compound1+[¹⁴C]-Compound 1)

Mean Actual Radioactive Dose: 5.09±0.05 MBq/kg

No clinical signs were observed following dosing.

Excretion.

Due to early terminal timepoints for animal 101 and 102, no excretacould be collected from these animals. Individual excretion data wasobtained from animal 103 and is presented TABLE 8. Cumulative data ofthe same animal is also presented in TABLE 8. Cumulative excretion isalso plotted in FIG. 1.

TABLE 8 Percentage of Administered Cumulative Percentage of DoseAdministered Dose Time (%) (%) Matrix (h) Animal 103 Urine 12 0.1 0.1 240.4 0.5 48 0.2 0.7 72 0.1 0.9 96 0.2 1.1 120 0.1 1.2 144 0.2 1.4 168 0.21.5 Subtotal 1.5 Feces 24 0.4 0.4 48 0.4 0.9 72 0.3 1.2 96 0.2 1.4 1200.1 1.5 144 0.1 1.7 168 0.1 1.7 Subtotal 1.7 Cage 24 0.1 0.1 Rinse 480.1 0.3 72 0.1 0.4 96 0.1 0.5 120 0.1 0.6 144 0.1 0.8 168 0.2 0.9Subtotal 0.9 TOTAL 168 4.2 4.2

TABLE 9 Concentration of Total Radioactivity Blood Plasma Animal TimeConcentration Concentration Blood to ID (h) (ng-eq/g) (ng-eq/g) PlasmaRatio 101 0.083 7826 11097 0.7 0.25 5497 8573 0.6 0.5 4134 6209 0.7 12913 4024 0.7 2 1752 2297 0.8 4 1256 1732 0.7 102 0.083 7615 11625 0.70.25 4998 7792 0.6 0.5 3818 5762 0.7 1 2755 3783 0.7 2 1651 2223 0.7 31396 1913 0.7 4 1299 1796 0.7 6 1217 1700 0.7 8 1205 1594 0.8 103 0.0837514 11547 0.7 0.25 4933 7783 0.6 0.5 3931 5914 0.7 1 2893 4166 0.7 21799 2427 0.7 3 1536 2077 0.7 4 1409 1900 0.7 6 1313 1881 0.7 8 12591710 0.7 12 1166 1625 0.7 24 1187 1525 0.8 168 1563 752 2.1

Following IV bolus administration of [¹⁴C]-Compound 1, a very limitedamount of administered radioactivity (4.2% of the dose) was excretedover 168 h post-dose, being most of the dose retained in the tissues:1.5% and 1.7% of the dose was recovered in urine and feces,respectively. Another 0.9% of the dose was recovered in the cage rinse,which relates to urinary excretion.

Blood and Plasma Pharmacokinetics

Individual concentrations of radioactivity in blood and plasma atvarious sampling times after a single IV bolus administration of[¹⁴C]-Compound 1 to male cynomolgus monkeys are presented in TABLE 9.Individual profiles of total radioactivity in blood and plasma aregraphically shown in FIG. 2 in Animal 101 (Panel A), Animal 102 (PanelB), Animal 103 over 24 hours (Panel C) and Animal 103 over 168 hours(Panel D).

PK parameters calculated from serial concentration time profiles oftotal radioactivity are presented in TABLE 10.

TABLE 10 Time C₀ C_(max) T_(max) T_(last) AUC_(0-t) ^(a) AUC_(0-inf)AUC_(Extrap) T_(1/2) Cl V_(z) V_(ss) Animal (h) Matrix (ng-eq/g)(ng-eq/g) (h) (h) (h · ng-eq/g) (h · ng-eq/g) (%) (h) mL/h/kg mL/kgmL/kg 101 4 Blood 9340 7830 0.08 4 10000 13400 25.1 1.85 374 1000 1020Plasma 12600 11100 0.08 4 14000 17900 21.3 1.52^(b) 280  615  670 102 8Blood 9390 7620 0.08 8 14400 5700 74.7 24.49^(b) 87.7 3100 2910 Plasma14200 11600 0.08 8 20300 73800 72.4 23.24^(b) 67.8 2270 2090 103 24Blood 9260 7510 0.08 24 34200 150000 77.2 67.76^(b) 33.3 3250 3210Plasma 14000 11500 0.08 24 47200 274000 82.8 103.05^(b) 18.3 2710 2640103 168 Blood 9260 7510 0.08 168 231000 747000 69.1 228.79^(b) 6.69 22102480 Plasma 14000 11500 0.08 168 205000 357000 42.7 140.69^(b) 14.0 28402780

Total drug-related radioactivity in blood and plasma was quantifiable upto the terminal timepoint in all animals.

In animal 103 (168 h post dose), systemic exposure to totalradioactivity, measured as AUC_(0-t), was 231000 and 205000 h·ng-eq/g inblood and plasma, respectively, while total radioactivity C_(max) was7510 ng-eq/g (C₀ 9260 ng-eq/g) and 11500 ng-eq/g (C₀ 14000 ng-eq/g),respectively.

CL was 6.69 mL/h/kg (blood) and 14.0 mL/h/kg (plasma) and wassignificantly lower than were hepatic and renal blood flow rates (whichare approximately 2616 and 1656 mL/h/kg). Consistently, mean V_(ss)accounting for 2480 mL/kg (blood) and 2780 mL/kg (plasma) wassignificantly greater than was total body water in cynomolgus monkey(which is approximately 693 mL/kg). This result indicated moderate (0.6to 5 L/kg) distribution into tissues.

The elimination phase could not be defined because of very limitedelimination over 168 h post-dose. Thus, acceptance criteria for terminalhalf-life T_(1/2) were not fully met and total radioactivity T_(1/2)could not be reliably calculated: estimates of 229 and 141 h in bloodand plasma, respectively.

Within 4 h of dosing (animal 101) a T_(1/2) of 1.85 h was evaluated inblood but indicated quick distribution rather than elimination.

Total radioactivity showed higher partitioning into plasma than blood(ratio 0.6-0.8) except for 168 h where a ratio of 2.1 was observed.

Tissues Distribution and Pharmacokinetics

Concentrations of radioactivity in tissues and correspondent tissue toblood ratios (T/B) obtained following single IV bolus administration of[¹⁴C]-Compound 1 to male cynomolgus monkeys at 5 mg/kg are presented inTABLE 11.

TABLE 11 Total DRM Concentration as ng-eq/g (Tissue to blood ratio)Animal 101 102 103 Tissue/Organ 4 h 12 h 168 h Adrenal gland_cortex10315 16063 8271 (8.2) (10.9) (5.0) Adrenal 5182 7779 8083 gland_medulla(4.1) (5.3) (4.9) Aortic wall 11777 9211 1954 (9.3) (6.3) (1.2)Bone_inner 1091 992 73.3 (0.9) (0.7) (0.04) Bone_surface 17582 6686 4995(13.9) (4.6) (3.0) Bone marrow 7487 13533 7478 (5.9) (9.2) (4.6)Brain_caudate 428 311 484 (0.3) (0.2) (0.3) Brain_cerebellum 493 349 378(0.4) (0.2) (0.2) Brain_cortex 471 440 424 (0.4) (0.3) (0.3)Brain_lateral 3763 3781 2868 ventricle (3.0) (2.6) (1.7) Brain_olfactorybulb 666 849 1360 (0.5) (0.6) (0.8) Brain_putamen 249 230 279 (0.2)(0.2) (0.2) Brain_substantia 526 151 NI nigra (0.4) (0.1) Brain_thalamus385 305 397 (0.3) (0.2) (0.2) Brown fat 2832 5324 2187 (2.2) (3.6) (1.3)Eye_uveal tract 2416 2366 1286 (choroid + RPE) (1.9) (1.6) (0.8) Hairfollicles 14769 15075 3628 (11.7) (10.3) (2.2) Heart_blood 1261 14681642 (1.0) (1.0) (1.0) Heart_myocardium 2908 4802 2517 (2.3) (3.3) (1.5)Joints (cartilage) 59612 130483 47762 (47.3) (88.9) (29.1) Kidney_cortex125167 145490 103160 (99.3) (99.1) (62.8) Kidney_medulla 3586 3198 1904(2.8) (2.2) (1.2) Large 3057 3282 476 intestine_content (2.4) (2.2)(0.3) Large 12369 13130 2690 intestine_mucosa (9.8) (8.9) (1.6) Largeintestine_wall 3088 4023 1296 (2.4) (2.7) (0.8) Liver 34695 56919 51575(27.5) (38.8) (31.4) Lung 1757 3243 1963 (1.4) (2.2) (1.2) Lymph nodes7256 14028 5755 (5.8) (9.6) (3.5) Pancreas 17587 9920 2595 (13.9) (6.8)(1.6) Pituitary gland 5581 5712 2850 (4.4) (3.9) (1.7) Salivary 1785419565 2790 glands_Parotid (14.2) (13.3) (1.7) Salivary glands_other15080 13356 2726 (12.0) (9.1) (1.7) Skeletal muscle 1425 1509 1235 (1.1)(1.0) (0.8) Skin 2585 2049 1228 (2.1) (1.4) (0.7) Small 3603 3520 603intestine_content (2.9) (2.4) (0.4) Small 10504 15425 2698intestine_mucosa (8.3) (10.5) (1.6) Small intestine_wall 3103 3655 1758(2.5) (2.5) (1.1) Spinal cord 484 401 412 (0.4) (0.3) (0.3) Spleen 83699236 7258 (6.6) (6.3) (4.4) Stomach_content 1858 2947 128 (0.6) (2.0)(0.1) Stomach_mucosa 9042 15960 3161 (7.2) (10.9) (1.9) Stomach_wall2975 2905 1764 (2.1) (2.0) (1.1) Testis 2363 5181 2630 (1.9) (3.5) (1.6)Thymus 5277 7601 3194 (4.2) (5.2) (1.9) Thyroid gland 1530 1793 2329(1.2) (1.2) (1.4) Urinary Bladder 3663 4350 1452 (2.9) (3.0) (0.9) Urine30379 4249 NP (24.1) (2.9) White fat 1162 NI NI (0.9) Whole blood^(a)1256 1205 1563 LOQ^(b) ≤47.1 ≤47.1 ≤43.0

Tissue concentrations and T/B values are also graphically presented inFIG. 3-6, respectively. FIG. 3 and FIG. 4 illustrate concentrations oftotal radioactivity in representative organs and tissues at varioustimes following single intravenous bolus of [¹⁴C]-Compound 1 to malecynomolgus monkeys at 5 mg/kg. FIG. 5 and FIG. 6 illustrate tissue toblood ratios at various times following single intravenous bolus ofradiocarbon-Compound 1 ([¹⁴C]-Compound 1) to male cynomolgus monkeys at5 mg/kg.

FIG. 7A, FIG. 7B, FIG. 8A, FIG. 8B, FIG. 9A, FIG. 9B, FIG. 10A, and FIG.10B, are autoradiographs that depict representative tissue distributionof total radioactivity in midsagittal parasagittal sections of Animal101 following a single intravenous 5 mg/kg bolus of [¹⁴C]-Compound 1.Any grey signal above background indicates presence of compound intissues. FIG. 11 is an autoradiograph that depicts representative tissuedistribution of total radioactivity in a mid-sagittal section of Animal101 following a single intravenous 5 mg/kg bolus of [¹⁴C]-Compound 1.Approximate concentrations of [¹⁴C]-Compound 1 in labelled regions ofFIG. 11 are as follows: 50 ng/g: 1 (Eye); 100 ng/g: 2 (Trachea), 3 (TA);210 ng/g: 4 (Stomach), 5 (Brain), 6 (Putamen), 7 (Caudate), 8(Substantia Nigra), 9 (Lateral Ventricle); 420 ng/g: 10 (Cortex), 11(Spiral cord); 870 ng/g: 12 (Skeletal muscle), 13 (Lung), 14 (Bladder),15 (Olfactory bulb), 16 (Cerebellum); 1600 ng/g: 17 (Kidney medulla), 18(Intestine), 19 (Heart (myocardium)), 20 (Spinal cord), 21 (Aorta), 22(Cardiac blood); 3600 ng/g: 23 (Mesenteric lymph nodes), 24 (Skin), 25(Pituitary); 7100 ng/g: 26 (Spleen), 27 (Adrenal gland), 28 (Pancreas),29 (Thymus); 14000 ng/g: 30 (Parotid), 31 (Bone marrow), 32 (Cartilage);29900 ng/g: 33 (Bone), 34 (Kidney Cortex), 35 (Cartilage (joint)), 36(Liver), 37 (Salivary glands), 38 (Lymph nodes).

FIG. 12A and FIG. 12B are autoradiographs that depict representativetissue distribution of total radioactivity in sagittal sections ofAnimal 101 (4 hours post dose), Animal 102 (12 hours post dose), Animal103 (7 days post dose) following a single intravenous 5 mg/kg bolus of[¹⁴C]-Compound 1. Any grey signal above background indicates presence ofcompound in tissues.

FIG. 13 are autoradiographs that depict representative cranial tissuedistribution of total radioactivity in selected sagittal sections ofAnimal 101 (4 hours post dose), Animal 102 (12 hours post dose), Animal103 (7 days post dose) following a single intravenous 5 mg/kg bolus of[¹⁴C]-Compound 1. Any grey signal above background indicates presence ofcompound in tissues.

FIG. 14 are autoradiographs that depict representative pelvic-areatissue distribution of total radioactivity in selected sagittal sectionsof Animal 101 (4 hours post dose), Animal 102 (12 hours post dose),Animal 103 (7 days post dose) following a single intravenous 5 mg/kgbolus of [¹⁴C]-Compound 1. Any grey signal above background indicatespresence of compound in tissues.

Within 4 hours of IV administration, radioactivity quickly and widelydistributed in all tissues investigated, with the vast majority oftissues containing concentrations higher than in blood (1261 ng-eq/g).

The highest tissue concentrations at 4 hours post-dose were observed inthe kidney cortex (125167 ng-eq/g, T/B 99.3), joints (likely connectedwith cartilage, 59612 ng-eq/g, T/B 47.3), and liver (34695 ng-eq/g, T/B27.5).

At this timepoint, several tissues showed a T/B>5: salivary glands(parotid T/B 14.2; others T/B 12.0), bone surface and pancreas (T/B13.9), hair follicles (T/B 11.7), large intestine mucosa (T/B 9.8),aortic wall (T/B 9.3), small intestine mucosa (T/B 8.3), adrenal cortex(T/B 8.2), stomach mucosa (T/B 7.2), spleen (T/B 6.6), bone marrow (T/B5.9), and lymph nodes (T/B 5.8).

At 12 h after dosing, concentrations were generally comparable to thoseobserved at the previous timepoint. At this timepoint, kidney cortex(145490 ng-eq/g, T/B 99.1), joints (130483 ng-eq/g, T/B 88.9), and liver(56919 ng-eq/g, T/B 38.8) were still the most exposed tissues andattained the highest concentration.

At this timepoint, several tissues showed a T/B≥5: salivary glands(parotid T/B 13.3; others T/B 9.1), pancreas (T/B 6.8), adrenal cortexand stomach mucosa (T/B 10.9), small intestine mucosa (T/B 10.5), hairfollicles (T/B 10.3), lymph nodes (T/B 9.6), bone marrow (T/B 9.2),large intestine mucosa (T/B 8.9), aortic wall and spleen (T/B 6.3),adrenal medulla (T/B 5.3), and thymus (T/B 5.2), compared to a cardiacblood concentration of 1468 ng-eq/g.

At 168 h post-dose, concentrations generally declined to concentrationscomparable to that in blood (1642 ng-eq/g). The highest concentrationswere observed in kidney cortex (103160 ng-eq/g, T/B 62.8), joints (47762ng-eq/g, T/B 88.9), and liver (51575 ng-eq/g, T/B 31.4). Theobservations suggested potential accumulation in these tissues.Moreover, at this timepoint, other tissues showed notable distribution(5<T/B<3): adrenal gland, bone marrow, spleen, and lymph nodes.

[¹⁴C]-Compound 1 showed moderate brain penetration (T/B<1) and appearedto distribute uniformly throughout the tissue and in all sub-regionsanalyzed (cerebellum, olfactory bulb, thalamus, caudate putamen,cerebral cortex, and substantia nigra). The highest concentrations weremeasured in the lateral ventricle instead: 3763, 3781, 2868 ng-eq/g at4, 12, and 168 h post-dose, respectively. This radioactivity was mostlikely connected to the choroid plexus within the ventricle.

[¹⁴C]-Compound 1 approximately equally partitioned between blood anduveal tract (choroid+retinal pigmented epithelium) in the eye (2416ng-eq/g (T/B 1.9), 2366 ng-eq/g (T/B 1.6), 1286 ng-eq/g (T/B 0.8) at 4,12, and 168 h post-dose, respectively). These observations indicatedpotential melanin binding. Consistently, high concentrations were alsomeasured in the hair follicles.

Conclusions

Following IV bolus administration of [¹⁴C]-Compound 1 to male Cynomolgusmonkeys only 4.2% of the administered dose was excreted over 168 hpost-dose: 1.5% of the dose was recovered in urine and 1.7% in feces.Cage rinse, which relates to urinary excretion, accounted for another0.9% of the dose.

Total drug-related radioactivity in blood and plasma was quantifiable upto the terminal timepoint in all animals.

Over 168 h post dose (Animal 103), systemic exposure to totalradioactivity, measured as AUC_(0-t), was 231000 and 205000 h·ng-eq/g inblood and plasma, respectively, while total radioactivity C_(max) was7510 ng-eq/g (C₀ 9260 ng-eq/g) and 11500 ng-eq/g (C₀ 14000 ng-eq/g),respectively.

[¹⁴C]-Compound 1 showed moderate (0.6 to 5 L/kg) volume of distributionand low clearance, thus the elimination half-life T_(1/2) could not bereliably calculated. T_(1/2) estimates were 229 and 141 h in blood andplasma, respectively.

Total radioactivity tended to partition into plasma (blood to plasmaratio 0.6-0.8) except for 168 h, where a ratio of 2.1 was observed.

In general, [¹⁴C]-Compound 1 drug-related material was widelydistributed throughout the whole body and quantifiable in all tissues upto the last timepoint sampled. The distribution pattern in tissues wascomparable between selected timepoints over 168 h post-dose. Very highconcentrations were observed in kidney cortex, joints (most likelyconnected to cartilage), and liver, suggesting potential accumulation inthese tissues.

Other tissues that showed notable uptake were salivary glands, bonesurface, pancreas, hair follicles, large intestine mucosa, aortic wall,small intestine mucosa, adrenal gland, stomach mucosa, spleen, bonemarrow, lymph nodes, and thymus.

Moderate brain penetration was observed (brain to blood ratio<1): totalradioactivity distributed quite uniformly in relevant sub-regions(cerebellum, olfactory bulb, thalamus, caudate putamen, cerebral cortex,and substantia nigra), but the highest concentrations were measured inthe lateral ventricle, most likely in the choroid plexus.

Finally, radioactivity was also measured in tissues containing melanin(e.g., hair follicles and the uveal tract in the eye), indicatingpotential binding to melanin.

Example 3. In Vivo Pharmacokinetic and Tissue Distribution StudyFollowing Single Intracerebroventricular Administration of Compound 1 inMale and Female C57BL6J Mouse

The objective of this study was to assess the pharmacokinetics andtissue distribution of Compound 1 in male and female C57BL6J micefollowing single intracerebroventricular (ICV) administration at 0.6, 1,and 2 mg/kg. One-hundred male and ninety-six female naïve C57BL6J micereceived Compound 1 at 0.3 mg/kg, (n=24 males and 32 females) or at 0.6mg/kg (n=32 males and 32 females) or at 1 mg/kg (n=32 males and 32females) or at 2 mg/kg (n=12 males).

One-hundred male and ninety-six female naïve C57BL6J mice receivedCompound 1 at 0.3 mg/kg, (n=24 males and 32 females) or at 0.6 mg/kg(n=32 males and 32 females) or at 1 mg/kg (n=32 males and 32 females) orat 2 mg/kg (n=12 males).

Due to severe clinical signs observed in males dosed at 2 mg/kg, thetreatment was stopped after the dosing of 12 males (4 for each timepointup to 8 hours). The dose of 2 mg/kg was replaced by a dose of 0.3 mg/kg.In the brain of animals given 2 mg/kg, the concentration at 8 hoursafter dosing was 3-fold higher than the concentration measured at thesame timepoint in animals given 1 mg/kg and 1.7-fold higher than C_(max)observed in males given 1 mg/kg.

Following a single ICV administration of Compound 1 at 0.3, 0.6, and 1mg/kg to male and female mice, Compound 1 was quantifiable up to 648hours after dosing (last collected PK timepoint) in the brain acrossdoses and in the heart at 0.3 mg/kg only in both sexes and up to atleast 312 hours after dosing in the spleen across doses in both sexes.The result indicated accumulation of test item in these tissues.Generally, Compound 1 was not quantifiable in plasma, intestine, liver,lung, kidney, and muscle across doses and in both sexes.

Maximum concentration occurred between 1.5 and 48 hours after dosing inbrain and between 4 and 48 hours in spleen in both sexes and across doserange evaluated. Maximum concentration occurred at 24 and 48 hours afterdosing in heart in females and males, respectively.

Mean composite C_(max) in the female and male brain was 3930 ng/mL and3110 ng/mL at 0.3 mg/kg, 11400 ng/mL and 6490 ng/mL at 0.6 mg/kg, and21500 ng/mL and 15500 ng/mL at 1 mg/kg, respectively. Mean compositeAUC_(last) in the female and male brain was 1800000 ng·h/mL and 1440000ng·h/mL at 0.3 mg/kg, 2360000 ng·h/mL and 2470000 ng·h/mL at 0.6 mg/kg,and 7150000 ng·h/mL and 5530000 ng·h/mL at 1 mg/kg, respectively. Meancomposite C_(max) in the female and male spleen was 586 ng/mL and 756ng/mL at 0.3 mg/kg, 984 ng/mL and 1530 ng/mL at 0.6 mg/kg, and 2270ng/mL and 2940 ng/mL at 1 mg/kg, respectively. Mean composite AUC_(last)in the female and male spleen was 96500 ng·h/mL and 48300 ng·h/mL at 0.3mg/kg, 98300 ng·h/mL and 166000 ng·h/mL at 0.6 mg/kg, and 449000 ng·h/mLand 1010000 ng·h/mL at 1 mg/kg, respectively.

Mean composite C_(max) in the female and male heart was 729 ng/mL and416 ng/mL and mean composite AUC_(last) in the female and male heart was184000 ng·h/mL and 160000 ng·h/mL, respectively.

Following ICV administration of Compound 1, for an increase in dose from0.3 to 1 mg/kg, the exposure (as mean composite C_(max) and AUC_(last))increased in brain in a proportional way as AUC_(last) and slightlysupra-proportionally as C_(max) and in spleen in a proportional way asmean composite C_(max) and supra-proportionally as mean composite AUC inboth sexes.

Generally, no notable (where notable is >2-fold) gender differences insystemic exposure in brain across dose range evaluated and in heart at0.3 mg/kg was observed, although C_(max) in heart was higher in femalesthan in males.

No notable gender differences in C_(max) in spleen was observed acrossdose range evaluated and in AUC at 0.3 mg/kg, whilst AUC was higher inmales than females at 0.6 and 1 mg/kg. This difference (as AUC₀₋₃₁₂) wasnotable at 0.6 mg/kg.

Following a single ICV administration of Compound 1 at 2 mg/kg to malemice, Compound 1 was quantifiable in the spleen and liver up to 8 hoursafter dosing (last collected PK timepoint), in the intestine, kidney,and lung only at 8 hours after dosing, in plasma only at 4 hour afterdosing. The compound was not quantifiable in muscle and heart in bothsexes.

Following a single ICV administration of Compound 1 at 0.3, 0.6, and 1mg/kg to male and female mice, Compound 1 was not quantifiable in urineand feces across doses and in both sexes.

Test and Control Items Test Item.

Compound 1

Purity: >90%

Storage Conditions: −80° C.

Vehicle.

DPBS: Dulbecco's Phosphate Buffered Saline (modified, without calciumchloride and magnesium chloride, liquid, sterile-filtered).

pH 7.1-7.5.

Osmolality: 275-304 mOs/kg.

Storage Conditions: Below 30° C. (Ambient temperature, AT), 2-8° C. whenopened.

Test Item Formulation.

Concentrations: 1.5 mg/mL, 3 mg/mL, 5 mg/mL, and 10 mg/mL (nominalconcentrations)

Method: The test item was dissolved in the vehicle. The formulation wasprepared dissolving the received amount of test item in the requiredamount of vehicle. A stock formulation at 10 mg/mL was prepared inadvance and divided in aliquots. Each aliquot was then diluted at thedifferent needed concentrations for each administration route. Thedilutions were prepared before the first day of administration for eachsession of administration/route. On the day of preparation of thedilutions, the stock formulation was thawed at AT and heated at approx.65° C. for 5 minutes, then allowed to cool to room temperature beforeuse. After stirring the formulation was used for the dilutions. Thediluted formulations were stored at AT (at least three days) inDispensary and used according to the scheduled time-points.

pH Range (measured only after the first preparation): 1.5 mg/mL: 6.75; 3mg/mL: 6.68; 5 mg/mL: 6.26; 10 mg/mL: 3.75.

Stability of the Stock at 10 mg/mL: at least 4 weeks at −80° C. and atleast 4 days at AT.

Stability of the diluted formulations: maximum 4 days at AT for thediluted formulations.

Residual Test Item Formulations: residual formulations were discarded atthe end of the dosing.

Test System

Mouse C57BL6J

Number on Study: 100 male and 96 female mice

Approximate Body Weight on Day 1: 20-25 gr

Type of Accommodation: Solid bottom plastic cages containing sawdustlitter.

Number per Cage: 4 of the same sex, treatment group, and timepoint percage.

Minimum Acclimatization: At least 5 days (prior to dosing).

Diet, Water, and Environmental Enrichment.

Diet Type: Rat and mouse maintenance diet Altromin-1324.

Water Source: Filtered from normal domestic supply.

Husbandry: Standard (as per internal Standard Operating Procedures).

Temperature Range: 20-22° C. (19-23° C. for less than 24 hoursacceptable).

Relative Humidity Range: 45-65% (40-70% for less than 24 hoursacceptable).

Lighting: Fluorescent lighting from approximately 06:00 to 18:00 hoursdaily.

Environmental Enrichment: 1 irradiated Iso-blox/5 animals, 1fun-tunnel/cage, and 1 mouse house/cage.

Experimental Design

The study design included 100 male and 96 female naïve C57BL6J mice andfour dose levels (0.3, 0.6, 1, and 2 mg/kg). Thirty-two males and 32females received each a single ICV administration of Compound 1 at 0.6and 1 mg/kg. Twelve males received each a single ICV administration ofCompound 1 at 2 mg/kg and twenty-four males and 32 females received eacha single ICV administration of Compound 1 at 0.3 mg/kg. Mice wereanaesthetized with isofluorane and the skin incised in the upper part ofthe skull to make the bregma visible. The injection into theintracerebral ventricle was performed using a microsyringe equipped witha 27G needle. At the end of the administration, the skin was sutured. Ananesthetic ointment was applied to the skin of the skull to limit thepain resulting from the incision. Dosing was performed in the presenceof a veterinary to observe whether any clinical signs were noted.

For each dose level four males (three males for group 4) and fourfemales were euthanized at the following timepoints: 1.5, 4, 8, 24hours, 3 days, 7 days, 14 days, and 28 days post-dosing.

From each animal, blood samples were collected through the cava vein ina composite scheme and at necropsy the following tissues were collected:brain, liver, lung, kidney, heart, small intestine, spleen, and skeletalmuscle.

In addition, the same animals that were euthanized at 24 hours, 3 days,7 days, 14 days, and 28 days post-dosing were placed in metabolic cagetwenty-four hours before euthanasia for collection of urine and feces inorder to collect 1 pooled (n=4, with the exception of males of Group 4for which n=3) urine and 1 pooled (n=4, with the exception of males ofGroup 4 for which n=3) fecal samples per timepoint/sex/dose level.

Methods and Procedures Formulation of Test Substance.

The dose formulation was administered by volume. The dose volumeadministered was 5 μL/mice. Formulations were maintained at ambient roomtemperature up to the end of the dosing procedure. The target doselevels are detailed in TABLE 12.

TABLE 12 Dose* Dose Animal Dose* Concentration Volume Group Sex Number(mg/kg) (mg/mL) (μL/mice) 1 M  1-32 0.6 3 5 F 33-64 2 M 65-96 1 5 5 F 97-128 3 M 129-140 2 10 5 4 M 141-160; 0.3 1.5 5 193-196 F 161-192*Expressed in terms of the parent compound

Dose Administration.

The dose formulation was administered via ICV route. Mice wereanaesthetized with isofluorane and the skin incised in the upper part ofthe skull to make the bregma visible. The injection into theintracerebral ventricle was performed using a microsyringe equipped witha 27G needle. At the end of the administration, the skin was sutured. Ananesthetic ointment was applied to the skin of the skull to limit thepain resulting from the incision.

No dose analysis was performed in the study; therefore, the nominaldoses are reported.

Sample Collection and Handling. Plasma.

After test item administration, terminal blood samples were collectedfrom cava vein of each animal at the following timepoints:

Day 1: 1.5 hour, 4 hours, 8 hours and 24 hours after dosing;Day 3 corresponding to 48 hours after dosing;Day 7 corresponding to 144 hours after dosing;Day 14 corresponding to 312 hours after dosing; andDay 28 corresponding to 648 hours after dosing.

Approximately 0.4-0.5 mL blood was collected into tubes containinganticoagulant (K₃EDTA), gently mixed and placed on crushed wet ice andthen spun by centrifuge (2000 g at +4° C. for 10 minutes) as soon aspracticable. The resultant plasma was separated from the erythrocytepellet and then transferred to uniquely labelled clear polypropylenetubes and frozen immediately over solid carbon dioxide or in a freezerat nominally −80° C.

Tissues.

After test item administration, brain including cerebellum, liver, lung,kidneys (both), heart, small intestine, spleen, and skeletal muscle werecollected from each animal at the following timepoints:

Day 1: 1.5 hour, 4 hours, 8 hours, and 24 hours after dosing;Day 3 corresponding to 48 hours after dosing;Day 7 corresponding to 144 hours after dosing;Day 14 corresponding to 312 hours after dosing; andDay 28 corresponding to 648 hours after dosing.

Gall bladder was removed from the liver before organ weight. Smallintestine was flushed with saline solution. Quadriceps femoris musclefrom both legs were collected.

Each tissue was weighed, and the weight was recorded. Tissue sampleswere rinsed in saline solution, transferred in polypropylene tubes orwrapped in aluminum foils and immediately frozen at −80° C.

Urine and Feces.

Urine and feces were obtained from 4 males (except for Group 4 for whichurine and feces were obtained from 3 males) and 4 females placed inmetabolic cages (food and water were left ad libitum) over 24 hoursbefore euthanasia at the following timepoints:

Day 1: 24 hours after dosing;Day 3 corresponding to 48 hours after dosing;Day 7 corresponding to 144 hours after dosing;Day 14 corresponding to 312 hours after dosing; andDay 28 corresponding to 648 hours after dosing.

Urine and feces were collected in plastic tubes and frozen over solidcarbon dioxide or in a freezer at −80° C. (nominal).

Storage of Samples.

All plasma samples were transported frozen and stored at nominally −80°C. until analyzed.

Sample Preparation and Analysis.

Samples were assayed for Compound 1 using a qualified method based onprotein precipitation followed by LC-MS/MS analysis.

Data Handling and Analysis

All computations utilized nominal sampling timepoints and nominal doses.

Individual data points at the same nominal time were averaged and then acomposite concentration-time profile was constructed for each sex ateach dose level. Pharmacokinetic (PK) analysis was performed on the meanconcentration-time profile and the resultant composite PK parameterswere reported.

The following parameters were reported:

-   -   Nominal dose levels (expressed as mg/kg).    -   Compound 1 plasma and tissue concentrations (expressed as        ng/mL).

Plasma and tissue PK elaboration were performed by non-compartmentalpharmacokinetics analysis using Phoenix WinNonlin. The followingparameters for test item, when feasible, were determined using thelinear logarithmic trapezoidal rule:

-   -   AUC_(last)=area under the plasma concentration time curve (AUC)        from the time of dosing to the last measurable concentration        (expressed as ng·h/mL)    -   C_(max)=maximum observed plasma concentration (expressed as        ng/mL)    -   T_(max)=time of maximum observed plasma concentration (expressed        as hours)    -   T_(last)=time of last measurable plasma concentration (expressed        as hours)    -   t_(1/2)=apparent terminal elimination half-life; t_(1/2) data        will be reported if r² was ≥0.9 (when rounded to one decimal        place) and the λ period [(λz_upper−λz_lower)/t½] values will be        >2.

Plasma concentrations and PK parameters are expressed in terms ofCompound as parent compound. Doses and temperatures are reported asnominal. The numerical data presented in this report are computergenerated.

Not quantifiable (NQ) values at early timepoints up were set to zero andincluded in the profiles, otherwise removed. Not quantifiable (NQ)values were treated as follows in the calculation of the composite meanconcentration values:

-   -   set to zero and included in the mean calculation: where the        resulting mean was below the LLQ, NQ was reported as mean value        for inclusion in the composite profile, if not, the numeric mean        value was reported.    -   if all results were NQ (or some were NA, NR, IS), NQ was        reported as mean value.

For the composite PK elaboration, mean NQ values were set to zero andincluded in the profiles at early timepoints but otherwise removed.

AUC_(last) and C_(max) values were reported with three significantdigits; T_(max) and T_(last) were displayed as the corresponding nominaltimes.

Results In Life Observations. Mortality/Morbidity.

Two females (No. 62; Group 1, 0.6 mg/kg and No. 120; Group 2, 1 mg/kg)and two males (Nos. 92 and 94; Group 2, 1 mg/kg) were found dead on Day6. Two males (Nos. 96 and 89; Group 2, 1 mg/kg) were found dead on Day12 and 13, respectively. Four females (Nos. 103, 106, 118, and 119;Group 2, 1 mg/kg) and two males (Nos. 136, 138; group 3, 2 mg/kg) wereeuthanized on Day 1 for human reasons. The animals showedhyperexcitability, locomotor ataxia, and convulsions.

One female (No. 125; Group 2; 1 mg/kg) was euthanized for human reasonon Day 9. The animal showed piloerection, significant weight loss, andlack of movement. Three females (No. 58; Group 1; 0.6 mg/kg; Nos. 122and 128; Group 2; 1 mg/kg) were euthanized for human reasons on Day 10.The animals showed piloerection, significant weight loss and lack ofmovement.

Seven males (Nos. 129, 130, 131, 132, 134, 135, and 140; Group 3, 2mg/kg) showed hyperexcitability, locomotor ataxia, tremors, acceleratedand jerky walking, and circular movements. These clinical signs wereobserved immediately after waking up from anesthesia and lasted untilthe time of scheduled necropsy (1.5, 4, and 8 hours, respectively). Onemale (No. 139; Group 3, 2 mg/kg) showed difficulty in waking up fromanesthesia and reacted slowly only when stimulated. The animal showedcircular movements to the left. Two males (Nos. 85 and 87; Group 2; 1mg/kg) showed reduced locomotor activity, hunched posture, and mildpiloerection. One male (No. 6, Group 1; 0.6 mg/kg) showed head tilted tothe right upon awakening.

Pharmacokinetics Analysis.

Summaries of plasma pharmacokinetic parameters in C57BL6J mice arepresented in TABLE 13, and tissue pharmacokinetic parameters in TABLE14.

TABLE 13 Dose T_(max) C_(max) AUC_(last) T_(last) t_(1/2) (mg/kg) (h)(ng/mL) (ng · h/mL) (h) (h) Males 0.3 NC NC NC NC NC 0.6 NC NC NC NC NC1 NC NC NC NC NC Females 0.3 NC NC NC NC NC 0.6 NC NC NC NC NC 1 NC NCNC NC NC NC = Not Calculable

TABLE 14 Brain Male Dose T_(max) C_(max) AUC_(last) T_(last) t_(1/2)(mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.3 1.5 3110 1440000 6482289.43* 0.6 4 6490 2470000 648 1989.19* 1 24 15500 5530000 648 1072.46*Female Dose T_(max) C_(max) AUC_(last) T_(last) t_(1/2) (mg/kg) (h)(ng/mL) (ng · h/mL) (h) (h) 0.3 48 3930 1800000 648 3085.79* 0.6 1.511400 2360000 648 4810.18* 1 8 21500 7150000 648 736.22* *Rsq < 0.9 andor Lambda factor < 2. The corresponding half-life is detailed forinformation only. Spleen Male Dose T_(max) C_(max) AUC_(last) T_(last)t_(1/2) (mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.3 4 756 48300 144111.42* 0.6 24 1530 166000 312 168.63* 1 48 2940 1010000 648 406.38*Female Dose T_(max) C_(max) AUC_(last) T_(last) t_(1/2) (mg/kg) (h)(ng/mL) (ng · h/mL) (h) (h) 0.3 8 586 96500 648 207.37 0.6 24 984 98300648 273.73* 1 48 2270 449000 312 NC *Rsq < 0.9 and or Lambda factor < 2.The corresponding half-life is detailed for information only NC = NotCalculable Heart Male Dose T_(max) C_(max) AUC_(last) T_(last) t_(1/2)(mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.3 48 416 160000 648 303.18*0.6 NC NC NC NC NC 1 NC NC NC NC NC Female Dose T_(max) C_(max)AUC_(last) T_(last) t_(1/2) (mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.324 729 184000 648 NC 0.6 NC NC NC NC NC 1 NC NC NC NC NC *Rsq < 0.9 andor Lambda factor < 2. The corresponding Half-life is detailed forinformation only NC = Not Calculable Liver Male Dose T_(max) C_(max)AUC_(last) T_(last) t_(1/2) (mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.3NC NC NC NC NC 0.6 NC NC NC NC NC 1 NC NC NC NC NC Female Dose T_(max)C_(max) AUC_(last) T_(last) t_(1/2) (mg/kg) (h) (ng/mL) (ng · h/mL) (h)(h) 0.3 NC NC NC NC NC 0.6 NC NC NC NC NC 1 NC NC NC NC NC NC = NotCalculable Kidney Male Dose T_(max) C_(max) AUC_(last) T_(last) t_(1/2)(mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.3 NC NC NC NC NC 0.6 NC NC NCNC NC 1 NC NC NC NC NC Female Dose T_(max) C_(max) AUC_(last) T_(last)t_(1/2) (mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.3 NC NC NC NC NC 0.6NC NC NC NC NC 1 NC NC NC NC NC NC = Not Calculable Intestine Male DoseT_(max) C_(max) AUC_(last) T_(last) t_(1/2) (mg/kg) (h) (ng/mL) (ng ·h/mL) (h) (h) 0.3 NC NC NC NC NC 0.6 NC NC NC NC NC 1 NC NC NC NC NCFemale Dose T_(max) C_(max) AUC_(last) T_(last) t_(1/2) (mg/kg) (h)(ng/mL) (ng · h/mL) (h) (h) 0.3 NC NC NC NC NC 0.6 NC NC NC NC NC 1 NCNC NC NC NC NC = Not Calculable Lung Male Dose T_(max) C_(max)AUC_(last) T_(last) t_(1/2) (mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.3NC NC NC NC NC 0.6 NC NC NC NC NC 1 NC NC NC NC NC Female Dose T_(max)C_(max) AUC_(last) T_(last) t_(1/2) (mg/kg) (h) (ng/mL) (ng · h/mL) (h)(h) 0.3 NC NC NC NC NC 0.6 NC NC NC NC NC 1 NC NC NC NC NC NC = NotCalculable Muscle Male Dose T_(max) C_(max) AUC_(last) T_(last) t_(1/2)(mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.3 NC NC NC NC NC 0.6 NC NC NCNC NC 1 NC NC NC NC NC Female Dose T_(max) C_(max) AUC_(last) T_(last)t_(1/2) (mg/kg) (h) (ng/mL) (ng · h/mL) (h) (h) 0.3 NC NC NC NC NC 0.6NC NC NC NC NC 1 NC NC NC NC NC NC = Not Calculable

The mean concentrations of Compound 1 in the brain samples are showngraphically in FIG. 15A for doses of 0.3 mg/kg, FIG. 15B for doses of0.6 mg/kg, FIG. 15C for doses of 1 mg/kg.

The mean concentrations of Compound 1 in the spleen samples are showngraphically in FIG. 16A for doses of 0.3 mg/kg, FIG. 16B for doses of0.6 mg/kg, FIG. 16C for doses of 1 mg/kg.

The mean concentrations of Compound 1 in the heart samples are showngraphically in FIG. 17 for doses of 0.3 mg/kg.

Summaries of plasma and tissue pharmacokinetic parameters in male andfemale C57BL6J mice are presented in TABLE 15.

TABLE 15 0.3 mg/kg 0.6 mg/kg 1 mg/kg Cmax AUClast Tmax Tlast CmaxAUClast Tmax Tlast Cmax AUClast Tmax Tlast Matrix (ng/mL) (ng · h/mL)(h) (h) (ng/mL) (ng · h/mL) (h) (h) (ng/mL) (ng · h/mL) (h) (h) Malesbrain 3110  1440000 1.5 648 6490 2470000  4 648 15500 5530000 24 648spleen 756  48300 4 144 1530  166000 24 312  2940 1010000 48 648 heart416  160000 48 648 NA NA NA NA NA NA NA NA liver NA NA NA NA NA NA NA NANA NA NA NA kidney NA NA NA NA NA NA NA NA NA NA NA NA intestine NA NANA NA NA NA NA NA NA NA NA NA lung NA NA NA NA NA NA NA NA NA NA NA NAmuscle NA NA NA NA NA NA NA NA NA NA NA NA plasma NA NA NA NA NA NA NANA NA NA NA NA Females brain 3930  1800000 48 648 11400   2360000 1.5648 21500 7150000  8 648 spleen 586  96500  8 648 984  98300 24 648 2270 449000 48 312 heart 729  184000 24 648 NA NA NA NA NA NA NA NAliver NA NA NA NA NA NA NA NA NA NA NA NA kidney NA NA NA NA NA NA NA NANA NA NA NA intestine NA NA NA NA NA NA NA NA NA NA NA NA lung NA NA NANA NA NA NA NA NA NA NA NA

Dose-proportionality ratios in brain and spleen is presented in TABLE16.

TABLE 16 Fold Difference in Exposure Male Female Fold Increase in MeanMean Mean Mean Ratio Dose C_(max) AUC_(last) C_(max) AUC_(last) Brain0.6/0.3 mg/kg 2.0 2.1 1.7 2.9 1.3   1/0.6 mg/kg 1.7 2.4 2.2 1.9 3.0  1/0.3 mg/kg 3.3 5.0 3.8 5.5 4.0 Spleen 0.6/0.3 mg/kg 2.0 2.0 2.4^(a)1.7 1.0   1/0.6 mg/kg 1.7 1.9 4.0^(b) 2.3 6.2^(b)   1/0.3 mg/kg 3.3 3.97.8^(a) 3.9 6.3^(b) ^(a)AUC144 bAUC312

Mean brain, spleen, and heart concentrations versus time profiles inmale and female C57BL6J mice are presented graphically in FIG. 18A fordoses of 0.3 mg/kg, FIG. 18B for doses of 0.6 mg/kg, FIG. 18C for dosesof 1 mg/kg.

Summaries of urine and fecal excretion are reported in TABLE 17.

TABLE 17 Dose (mg/kg) Time 0.3 0.6 1 (h) Males Females Males FemalesMales Females Urine  24 NC NC NC NC NC NC  48 NC NC NC NC NC NC 144 NCNC NC NC NC NC 312 NC NC NC NC NC NC 648 NC NC NC NC NC NC Feces  24 NCNC NC NC NC NC  48 NC NC NC NC NC NC 144 NC NC NC NC NC NC 312 NC NC NCNC NC NC 648 NC NC NC NC NC NC NC = Not calculable

Discussion Brain

Following a single ICV administration of Compound 1 at 0.3, 0.6, and 1mg/kg to male and female mice, Compound 1 was quantifiable in the brainup to 648 hours after dosing (last collected PK timepoint) across dosesand in both sexes. T_(max) ranged between 1.5 and 48 hours after dosingin both sexes across dose range evaluated. Mean composite C_(max) in thefemale and male brain was 3930 ng/mL and 3110 ng/mL at 0.3 mg/kg, 11400ng/mL and 6490 ng/mL at 0.6 mg/kg, and 21500 ng/mL and 15500 ng/mL at 1mg/kg, respectively. Mean composite AUC_(last) in the female and malebrain was 1800000 ng·h/mL and 1440000 ng·h/mL at 0.3 mg/kg, 2360000ng·h/mL and 2470000 ng·h/mL at 0.6 mg/kg, and 7150000 ng·h/mL and5530000 ng·h/mL at 1 mg/kg, respectively.

Following intracerebroventricular administration of Compound 1, theexposure (as mean composite C_(max) and AUC_(last)) in brain increasedwith increasing dose from 0.3 to 1 mg/kg in a proportional way asAUC_(last) and slightly supra-proportionally as C_(max) in both sexes.

Generally, no notable (where notable is >2-fold) gender differences insystemic exposure in brain were observed across dose range evaluated.

In the brain of animals given 2 mg/kg the concentration at 8 hours afterdosing was 3-fold higher than the concentration measured at the sametimepoint in animals given 1 mg/kg and 1.7-fold higher than C_(max)observed in males given 1 mg/kg.

Spleen

Following a single ICV administration of Compound 1 at 0.3, 0.6, and 1mg/kg to male and female mice, Compound 1 was quantifiable in the spleenup to 648 hours after dosing (last collected PK timepoint) in females at0.3 and 0.6 mg/kg and in males at 1 mg/kg, up to 312 hours in males at0.6 mg/kg and in females at 1 mg/kg and up to 144 hours in males at 0.3mg/kg. T_(max) ranged between 4 and 48 hours after dosing in both sexesacross the dose range evaluated. Mean composite C_(max) in the femaleand male spleen was 586 ng/mL and 756 ng/mL at 0.3 mg/kg, 984 ng/mL and1530 ng/mL at 0.6 mg/kg, and 2270 ng/mL and 2940 ng/mL at 1 mg/kg,respectively. Mean composite AUC_(last) in the female and male spleenwas 96500 ng·h/mL and 48300 ng·h/mL at 0.3 mg/kg, 98300 ng·h/mL and166000 ng·h/mL at 0.6 mg/kg, and 449000 ng·h/mL and 1010000 ng·h/mL at 1mg/kg, respectively.

Where calculable (in females dosed at 0.3 mg/kg/day), t_(1/2) was 208hours.

Following intracerebroventricular administration of Compound 1, theexposure in spleen increased with increasing dose from 0.3 to 1 mg/kg ina proportional way as mean composite C_(max) and supra-proportionally asmean composite AUC in both sexes.

Generally, no notable (where notable is >2-fold) gender differences inC_(max) in spleen were observed across dose range evaluated and in AUCat 0.3 mg/kg, while AUC was higher in males than females at 0.6 and 1mg/kg. This difference (as AUC0-312) was notable at 0.6 mg/kg.

Following a single intracerebroventricular administration of Compound 1at 2 mg/kg to male mice, Compound 1 was quantifiable in the spleen up to8 hours after dosing (last collected PK timepoint), with concentrations1.4-fold higher than those observed at the same timepoints in malesgiven 1 mg/kg.

Heart.

Following a single intracerebroventricular administration of Compound 1at 0.3, 0.6, and 1 mg/kg to male and female mice, Compound 1 wasquantifiable in the heart up to 648 hours after dosing (last collectedPK timepoint) only at 0.3 mg/kg and up to 4 hours after dosing at 0.6mg/kg in both sexes. T_(max) occurred at 24 and 48 hours after dosing infemales and males, respectively. Mean composite C_(max) in the femaleand male heart was 729 ng/mL and 416 ng/mL. Mean composite AUC_(last) inthe female and male heart was 184000 ng·h/mL and 160000 ng·h/mL.Generally, no notable (where notable is >2-fold) gender differences insystemic exposure in heart was observed, although C_(max) was higher infemales than in males. Following a single intracerebroventricularadministration of Compound 1 at 2 mg/kg to male mice, Compound 1 was notquantifiable in the heart.

Plasma.

Following a single intracerebroventricular administration of Compound 1at 0.3, 0.6, and 1 mg/kg to male and female mice, Compound 1 was notquantifiable in plasma at all timepoints across doses and in both sexes.

Following a single intracerebroventricular administration of Compound 1at 2 mg/kg to male mice, Compound 1 was quantifiable in plasma only at 4hour after dosing.

Liver.

Following a single ICV administration of Compound 1 at 0.3, 0.6, and 1mg/kg to male and female mice, generally Compound 1 was not quantifiablein the liver across dose range and in both sexes.

Following a single ICV administration of Compound 1 at 2 mg/kg in malemice, Compound 1 was quantifiable in liver up to 8 hours after dosing(last point of tissue collection).

Kidney.

Following a single intracerebroventricular administration of Compound 1at 0.3, 0.6, and 1 mg/kg to male and female mice, Compound 1 was notquantifiable in kidney across doses and in both sexes.

Following a single intracerebroventricular administration of Compound 1at 2 mg/kg to male mice, Compound 1 was quantifiable in kidney only at 8hours after dosing (last point of tissue collection).

Intestine.

Following a single intracerebroventricular administration of Compound 1at 0.3, 0.6, and 1 mg/kg to male and female mice, generally Compound 1was not quantifiable in the intestine across doses and in both sexes.

Following a single ICV administration of Compound 1 at 2 mg/kg to maleand female mice, Compound 1 was quantifiable in the intestine only at 8hours after dosing.

Lung.

Following a single intracerebroventricular administration of Compound 1at 0.3, 0.6, and 1 mg/kg to male and female mice, Compound 1 was notquantifiable in the lung across dose range and in both sexes.

Following a single intracerebroventricular administration of Compound 1at 2 mg/kg to male mice, Compound 1 was quantifiable in lung only at 8hours after dosing (last point of tissue collection).

Muscle.

Following a single ICV administration of Compound 1 at 0.3, 0.6, and 1mg/kg to male and female mice and at 2 mg/kg to male mice, generallyCompound 1 was not quantifiable in muscle across dose range and in bothsexes.

Urine.

Following a single ICV administration of Compound 1 at 0.3, 0.6, and 1mg/kg to male and female mice, Compound 1 was not quantifiable in theurine across doses and in both sexes.

Feces.

Following a single intracerebroventricular administration of Compound 1at 0.3, 0.6, and 1 mg/kg to male and female mice, Compound 1 was notquantifiable in the feces across doses and in both sexes.

Conclusions

Following a single ICV administration of Compound 1 at 0.3, 0.6, and 1mg/kg to male and female mice, Compound 1 was quantifiable up to 648hours after dosing (last collected PK timepoint) in the brain acrossdoses and in the heart only at 0.3 mg/kg in both sexes and up to atleast 312 hours after dosing in the spleen across doses in both sexes.The result indicates accumulation of test item in these tissues.Generally, Compound 1 was not quantifiable in plasma, intestine, liver,lung, kidney, and muscle across doses and in both sexes.

Maximum concentration of Compound 1 occurred between 1.5 and 48 hoursafter dosing in brain and between 4 and 48 hours in spleen and in bothsexes and across dose range evaluated and at 24 and 48 hours afterdosing in heart in females and males, respectively.

Following ICV administration of Compound 1, for an increase in dose from0.3 to 1 mg/kg the exposure (as mean composite C_(max) and AUC_(last))increased in brain in a proportional way as AUC_(last) and slightlysupra-proportionally as C_(max) and in spleen in a proportional way asmean composite C_(max) and supra-proportionally as mean composite AUC inboth sexes.

Generally, no notable (where notable is >2-fold) gender differences insystemic exposure in brain across dose range evaluated and in heart wasobserved, although C_(max) in heart was higher in females than in males.

No notable gender differences in C_(max) in spleen was observed acrossdose range evaluated and in AUC at 0.3 mg/kg, while AUC was higher inmales than females at 0.6 and 1 mg/kg. This difference (as AUC₀₋₃₁₂) wasnotable at 0.6 mg/kg.

Following a single ICV administration of Compound 1 at 2 mg/kg to malemice, Compound 1 was quantifiable in the spleen and liver up to 8 hoursafter dosing (last collected PK timepoint) and in the intestine, kidneyand lung only at 8 hours after dosing, in plasma only at 4 hour afterdosing. The compound was not quantifiable in muscle and heart in bothsexes.

Following a single ICV administration of Compound 1 at 0.3, 0.6, and 1mg/kg to male and female mice, Compound 1 was not quantifiable in urineand feces across doses and in both sexes.

Example 4. Knockdown of Mutant Huntington's Disease Protein by Compoundsof the Disclosure

This example demonstrates the ability of illustrative compounds of thedisclosure to reduce selectively and potently expression of mutantHuntington's disease protein in cells without causing cytotoxicity.

A human subject-derived fibroblast cell line GM09197 was obtained fromthe Coriell Institute (Coriell Institute for Medical Research, Camden,N.J.). The first huntingtin allele in this cell line is a mutanthuntingtin allele with 151 CAG repeats (mHTT), which is associated withdevelopment of Huntington's disease. The second huntingtin allele inthis cell line contains 21 CAG repeats. In some embodiments, this levelcan be considered a normal or wild type huntingtin allele (wtHTT) thatis not considered to cause Huntington's disease. Cells were maintainedat 37° C. and 5% CO₂ in minimal essential media (MEM) supplemented withnon-essential amino acids (Corning Inc, Corning N.Y., cat. n #10-009-CV)and 10% heat inactivated FBS (Corning Inc., Corning N.J. cat. n#35-016-CV).

Peptide nucleic acid (PNA) compounds of the disclosure were screened fortoxicity and the ability to knockdown expression selectively of mHTT.Cells were plated in 24-well plates at 150,000 cells/well insupplemented MEM one day before addition of PNA. Stock solutions of PNAwere heated at 80° C. for 10 min before use and were added to finalconcentrations of 1 μM or 5 μM in the cell cultures. Cells wereincubated for 3 days in the presence or absence of PNA compound beforeevaluation of huntingtin knockdown and cytotoxicity.

For huntingtin knockdown assays, cells were harvested with atrypsin-EDTA solution (0.25%, Invitrogen, Carlsbad, Calif., cat. n#25200072) and lysed using M-Per buffer (Thermo Fischer Scientific,Waltham, Mass., cat. n #78503). Total protein present in the lysateswere quantified using BCA assay (Pierce BCA protein Assay Kit cat. n#23225, Thermo Fischer Scientific, Waltham, Mass.). mHTT and wtHTTproteins were separated by SDS-PAGE using 4-15% gradient gels (Bio-RadLaboratories, Hercules, Calif., cat. n #4561083) with Tris GlycineRunning Buffer 0.1% SDS pH8.3 (Glycine cat. n #G8898, Sigma-Aldrich, StLouis Mo., Tri-Base cat. n #BP152-500, Fisher Scientific, Hampton, N.H.,SDS 10% solution cat. n #1610416, Bio-Rad Laboratories, Hercules,Calif.). Gels were run at 165V for 2 hours. After gel electrophoresis,proteins were transferred to a nitrocellulose membrane (Bio-RadLaboratories, Hercules, Calif., cat. n #1620112).

A huntingtin-specific primary antibody was used to bind specificallyboth mHTT and wtHTT (1:2500, Abcam, Cambridge UK, cat. n #ab109115),with a beta-actin-specific primary antibody as a control (1:5000, AbcamCambridge, UK, cat. n #ab8227). Horseradish peroxidase (HRP) conjugatedanti-mouse or anti-rabbit secondary antibodies (1:10,000, JacksonImmunoResearch Laboratories, West Grove, Pa., cat. n #315-035-0003 and111-035-045) were used for visualizing proteins using SuperSignal WestPico Plus Chemiluminescent Substrate (Thermo Fischer Scientific,Waltham, Mass., cat. n #34577). Protein bands were quantified usingiBright Analysis Software (Thermo Fischer Scientific, Waltham, Mass.).mHTT and wtHTT bands were normalized according to beta-actin expression,and mHTT and wtHTT expression inhibition was calculated as a relativevalue to untreated control cells.

FIG. 19A shows the relative expression of wtHTT (darker bars) and mHTT(lighter bars) after treatment with Compound 2 at 1 or 5 μM. The figuredemonstrates knockdown of both mHTT and wtHTT at 5 μM, with somewhatgreater knockdown of mHTT than wtHTT.

FIG. 19B shows the relative expression of wtHTT (darker bars) and mHTT(lighter bars) after treatment with Compound 3 at 1 or 5 μM. The figuredemonstrates knockdown of mHTT and wtHTT, with higher selectivity formHTT compared to wtHTT at 1 μM. These results also show that Compound 3exhibited greater potency than Compound 2 for mHTT knockdown in thisassay.

FIG. 19C shows the relative expression of wtHTT (darker bars) and mHTT(lighter bars, not visible) after treatment with Compound 4 at 1 or 5The mHTT bars are not visible as mHTT was below the limit of detection.These results demonstrate that Compound 4 exhibits higher potency andselectivity for knocking down mHTT compared to Compound 3 and Compound 2in this assay.

These results demonstrate a structure-activity relationship betweenillustrative PNA compounds of the disclosure and potency and selectivityfor modulating expression of target genes and proteins. As the PNAcompounds evaluated comprise backbone modifications that alter theirbinding affinity for mHTT transcript, these results also show thatmodulating binding affinity via PNA backbone modifications can altertarget selectivity and therapeutic potency.

To evaluate potential toxicity of the PNA compounds in the assayconditions, a Lactate Dehydrogenase (LDH) release cytotoxicity assay wasperformed using a CyQuant LDH kit (cat. n #C20300, Invitrogen, Carlsbad,Calif.,).

For a maximum LDH release control, 10× Lysis Buffer was added to controlwells comprising the GM09197 cells, and the plate was incubated at 37°C., 5% CO₂ for 50 min.

50 μL of medium from each sample and control well was transferredcontrols to designated wells in a 96-well flat bottom plate. An LDHPositive Control from the kit was also used, as were basal medium LDHactivity controls, with 50 μL of culture medium±serum per basal LDHcontrol well, and an untreated (PBS) control.

50 μL of Reaction Mixture was transferred to each sample and controlwell and mixed by gentle tapping. The plate was incubated at roomtemperature for 30 min protected from light, after which 50 μL of StopSolution to was added to each well. Absorbance at 490 nm and 680 nm wasmeasured. To determine LDH activity, the 680-nm absorbance value(background signal from instrument) was subtracted from the 490-nmabsorbance value.

The percent of dead cells detected following treatment with the PNAcompounds was not significantly different than that of untreatedcontrol. The result indicates that cytotoxicity was not observed forGM09197 cells treated with PNA compounds of the disclosure Compound 2(FIG. 20A), Compound 3 (FIG. 20B), and Compound 4 (FIG. 20C) at dosesthat were effective to knock down mHTT.

Example 5. In Vivo Tolerability of PNA Compounds

This example demonstrates that illustrative PNA compounds of thedisclosure can be well tolerated when administered to subjects.

PNA compounds of the disclosure were administered to non-human primatesintravenously via a single tail vein injection per animal. The singledose injections were well tolerated at doses of up to 5 mg/kg.

PNA compounds of the disclosure were administered via intraperitonealinjection into mice three times per week. Three times weekly doses of upto 2 mg/kg were well tolerated for up to five weeks.

Embodiments

The following non-limiting embodiments provide illustrative examples ofthe invention, but do not limit the scope of the invention.

Embodiment 1. A compound comprising a chain, wherein the chain comprisesa series of atoms concatenated to form the chain, wherein a plurality ofthe atoms that are concatenated to form the chain are each independentlysubstituted with a substituent that bears a guanidino group, wherein thechain has a pattern of one atom that is independently substituted with asubstituent that bears a guanidino group, followed by five consecutiveatoms that are not substituted by a substituent that bears a guanidinogroup, followed by a second atom that is independently substituted witha substituent that bears a guanidino group, followed by another fiveconsecutive atoms that are not substituted by a substituent that bears aguanidino group, followed by a third atom that is independentlysubstituted with a substituent that bears a guanidino group, wherein afirst end of the chain or a second end of the chain is substituted witha peptide.

Embodiment 2. The compound of embodiment 1, wherein the pattern furthercomprises one atom that is independently substituted with a substituentthat bears a first nucleobase, followed by five consecutive atoms thatare not substituted by a substituent that bears a nucleobase, followedby a second atom that is independently substituted with a substituentthat bears a second nucleobase, followed by another five consecutiveatoms that are not substituted by a substituent that bears a nucleobase,followed by a third atom that is independently substituted with asubstituent that bears a third nucleobase.

Embodiment 3. The compound of embodiment 1 or embodiment 2, wherein eachsubstituent that bears a guanidino group is independentlyguanidinoalkylene.

Embodiment 4. The compound of embodiment 1 or embodiment 2, wherein eachsubstituent that bears a guanidino group is 3-guanidino-prop-1-yl.

Embodiment 5. The compound of embodiment 1 or embodiment 2, wherein eachsubstituent that bears a guanidino group is 4-guanidino-but-1-yl.

Embodiment 6. The compound of embodiment 2, wherein the substituent thatbears the first nucleobase, the substituent that bears the secondnucleobase, and the substituent that bears the third nucleobase are eachindependently purinylacyl, purinylalkylene, pyrimidinylacyl, orpyrimidinylalkylene.

Embodiment 7. The compound of embodiment 2, wherein the substituent thatbears the first nucleobase, the substituent that bears the secondnucleobase, and the substituent that bears the third nucleobase are eachindependently guaninylacyl, adeninylacyl, cytosinylacyl, thyminylacyl,or uracilylacyl.

Embodiment 8. The compound of embodiment 2, wherein the firstnucleobase, the second nucleobase, and the third nucleobase form asequence that is CTG, TGC, or GCT.

Embodiment 9. The compound of any one of embodiments 1-8, wherein thecompound is a peptide nucleic acid oligomer.

Embodiment 10. The compound of any one of embodiments 1-8, wherein thecompound is a gamma peptide nucleic acid oligomer.

Embodiment 11. The compound of any one of embodiments 1-8, wherein thecompound is a peptide nucleic acid oligomer, and the peptide nucleicacid oligomer comprises a series of nucleobase side chains that form asequence that is (CTG)_(n), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or10.

Embodiment 12. The compound of any one of embodiments 1-11, wherein thecompound is a peptide nucleic acid oligomer, wherein the first end ofthe chain is an N-terminus of the peptide nucleic acid oligomer, and thesecond end of the chain is a C-terminus of the peptide nucleic acidoligomer.

Embodiment 13. The compound of embodiment 12, wherein the C-terminus ofthe peptide nucleic acid oligomer is bound by a peptide bond to thepeptide.

Embodiment 14. The compound of embodiment 12, wherein the C-terminus ofthe peptide nucleic acid oligomer is bound by a peptide bond to anamidated lysine residue.

Embodiment 15. The compound of any one of embodiments 12-14, wherein anN-terminus of the peptide nucleic acid oligomer is unsubstituted.

Embodiment 16. The compound of any one of embodiments 12-14, wherein anN-terminus of the peptide nucleic acid oligomer is bound by a peptidebond to a peptide.

Embodiment 17. A compound comprising a peptide nucleic acid sequence anda cell permeabilizing group attached to the peptide nucleic acidsequence, wherein if a radiolabeled analogue of the compound issubjected to an assay, wherein the assay comprises:

-   -   (a) a first component, wherein the first component comprises:        -   (i) administering a 5 mg/kg intravenous bolus dose of the            radiolabeled analogue to a caudal vein of a monkey, wherein            the monkey is a male Cynomolgus monkey;        -   (ii) euthanizing the monkey 4 hours after the administering;        -   (iii) after (ii), freezing the monkey in a mixture of hexane            and solid carbon dioxide for at least two hours to provide a            frozen carcass;        -   (iv) embedding the frozen carcass, left lateral side            uppermost, in 2% w/v aqueous sodium carboxymethylcellulose            to provide an embedded carcass;        -   (v) sectioning the embedded carcass into 40 μm sagittal            whole body sections with a cryomacrotome;        -   (vi) mounting the 40 μm sagittal whole body sections on            pressure sensitive tape;        -   (vii) after (vi), dehydrating the whole body sections in the            cryomacrotome at about −20° C. for about 60 hours;        -   (viii) after (vii), placing the whole body sections against            an image plate sensitive to carbon-14 for no longer than            four days;        -   (ix) after (viii), scanning the image plate with a phosphor            imager system; and        -   (x) after (ix), determining a concentration of the            radiolabeled analogue in brain tissue of the whole body            sections; and    -   (b) a second component, wherein the second component is        analogous to the first component except that the second        component uses another monkey that is euthanized 168 hours after        the administering,    -   wherein the radiolabeled analogue comprises an N-terminus that        is substituted with a ¹⁴C-enriched glycine residue, and the        radiolabeled analogue consists of a structure that differs from        the compound solely in that the compound lacks the ¹⁴C-enriched        glycine residue of the radiolabeled analogue,    -   then in the assay, the concentration of the radiolabeled        analogue in brain tissue determined in the second component is        equivalent to at least about 80% of the concentration of the        radiolabeled analogue in brain tissue determined in the first        component.

Embodiment 18. The compound of embodiment 17, wherein the brain tissueis cortex tissue, caudate nucleus tissue, olfactory bulb tissue, putamentissue, or thalamus tissue.

Embodiment 19. The compound of embodiment 17, wherein the brain tissueis cortex tissue, caudate nucleus tissue, olfactory bulb tissue, putamentissue, and thalamus tissue.

Embodiment 20. The compound of embodiment 17, wherein in the assay, theconcentration of the radiolabeled analogue in brain tissue determined inthe second component is equivalent to at least about 100% of theconcentration of the radiolabeled analogue in brain tissue determined inthe first component.

Embodiment 21. The compound of embodiment 20, wherein the brain tissueof the sections is caudate nucleus tissue, olfactory bulb tissue,putamen tissue, or thalamus tissue.

Embodiment 22. The compound of embodiment 20, wherein the brain tissueof the sections is caudate nucleus tissue, olfactory bulb tissue,putamen tissue, and thalamus tissue.

Embodiment 23. The compound of embodiment 17, wherein in the assay, theconcentration of the radiolabeled analogue in brain tissue determined inthe second component is equivalent to at least about 150% of theconcentration of the radiolabeled analogue in brain tissue determined inthe first component.

Embodiment 24. The compound of embodiment 23, wherein the brain tissueof the sections is olfactory bulb tissue.

Embodiment 25. The compound of any one of embodiments 17-24, wherein theassay further comprises a third component, wherein the third componentis analogous to the first component except that the second componentuses another monkey that is euthanized 12 hours after the administering,wherein in the assay, the concentration of the radiolabeled analogue inbrain tissue determined in the third component is equivalent to at leastabout 80% of the concentration of the radiolabeled analogue in braintissue determined in the first component.

Embodiment 26. The compound of embodiment 25, wherein the brain tissueof the sections is cortex tissue, olfactory bulb tissue, putamen tissue,lateral ventricle tissue, or thalamus tissue.

Embodiment 27. The compound of embodiment 25, wherein the brain tissueof the sections is cortex tissue, olfactory bulb tissue, putamen tissue,lateral ventricle tissue, and thalamus tissue.

Embodiment 28. The compound of embodiment 25, wherein in the assay, theconcentration of the radiolabeled analogue in brain tissue determined inthe third component is equivalent to at least about 100% of theconcentration of the radiolabeled analogue in brain tissue determined inthe first component.

Embodiment 29. The compound of embodiment 28, wherein the brain tissueis olfactory bulb tissue or lateral ventricle tissue.

Embodiment 30. The compound of embodiment 28, wherein the brain tissueis olfactory bulb tissue and lateral ventricle tissue.

Embodiment 31. The compound of any one of embodiments 17-30, wherein thecell permeabilizing group is an alpha substituent of the peptide nucleicacid.

Embodiment 32. The compound of any one of embodiments 17-30, wherein thecompound is a gamma peptide nucleic acid.

Embodiment 33. The compound of embodiment 32, wherein the cellpermeabilizing group is a gamma substituent of the gamma peptide nucleicacid.

Embodiment 34. The compound of embodiment 33, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are eachindependently guanidinoalkylene.

Embodiment 35. The compound of embodiment 33, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are each3-guanidino-prop-1-yl.

Embodiment 36. The compound of embodiment 33, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are each4-guanidino-but-1-yl.

Embodiment 37. The compound of any one of embodiments 17-36, wherein thepeptide nucleic acid sequence is (CTG)_(n), wherein n is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10.

Embodiment 38. The compound of any one of embodiments 17-37, wherein aC-terminus of the peptide nucleic acid is bound by a peptide bond to apeptide.

Embodiment 39. The compound of any one of embodiments 17-37, wherein aC-terminus of the peptide nucleic acid is bound by a peptide bond to anamidated lysine residue.

Embodiment 40. The compound of any one of embodiments 17-39, wherein anN-terminus of the peptide nucleic acid of the compound is unsubstituted.

Embodiment 41. A compound comprising a peptide nucleic acid sequence,wherein the peptide nucleic acid sequence comprises: (i) a series ofpeptide nucleic acid residues having a repeating triad of nucleobaseside chains; and (ii) a cell permeabilizing group attached to the seriesof peptide nucleic acid residues, wherein if the compound is subjectedto an assay, and the assay comprises:

-   -   (a) administering by intracerebroventricular administration a        dose amount of about 0.1 mg/kg to about 2 mg/kg of the compound        to mice;    -   (b) euthanizing the mice at a time point between about 1 hour        and 28 days post intracerebroventricular administration;    -   (c) collecting brain tissues from the mice after the        euthanizing; and    -   (d) using liquid chromatography-tandem mass spectrometry to        determine concentrations of the brain tissues in the mice, then        in the assay, a mean maximum brain concentration is observed in        the mice at a time to maximum brain concentration of about 1        hour to about 50 hours post administration and the mean maximum        brain concentration of the mice is observed to be about 3000        ng/mL to about 22000 ng/mL.

Embodiment 42. The compound of embodiment 41, wherein the compound is agamma peptide nucleic acid.

Embodiment 43. The compound of embodiment 42, wherein the cellpermeabilizing group is an alpha substituent of the gamma peptidenucleic acid.

Embodiment 44. The compound of embodiment 42, wherein the cellpermeabilizing group is a gamma substituent of the gamma peptide nucleicacid.

Embodiment 45. The compound of embodiment 43, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are eachindependently guanidinoalkylene.

Embodiment 46. The compound of embodiment 43, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are each3-guanidino-prop-1-yl.

Embodiment 47. The compound of embodiment 43, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are each4-guanidino-but-1-yl.

Embodiment 48. The compound of any one of embodiments 41-47, wherein thepeptide nucleic acid sequence is (CTG)_(n), wherein n is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10.

Embodiment 49. The compound of any one of embodiments 41-48, wherein aC-terminus of the peptide nucleic acid sequence is bound by a peptidebond to a peptide.

Embodiment 50. The compound of any one of embodiments 41-48, wherein theC-terminus of the peptide nucleic acid sequence is bound by a peptidebond to an amidated lysine residue.

Embodiment 51. The compound of any one of embodiments 41-50, wherein anN-terminus of the peptide nucleic acid sequence of the compound isunsubstituted.

Embodiment 52. The compound of any one of embodiments 41-51, wherein if(1) the mice are male mice, the mean maximum brain concentration of themale mice is observed to be about 3110 ng/mL at the time to maximumbrain concentration of about 1.5 hours and the dose amount of about 0.3mg/kg, the mean maximum brain concentration of the male mice is observedto be about 6490 ng/mL at the time to maximum brain concentration ofabout 4 hours and the dose amount of about 0.6 mg/kg, and the meanmaximum brain concentration of the male mice is observed to be about15500 ng/mL at the time to maximum brain concentration of about 24 hoursand the dose amount of about 1 mg/kg; and (2) if the mice are femalemice, the mean maximum brain concentration of the female mice isobserved to be about 3930 ng/mL at the time to maximum brainconcentration of about 48 hours and a dose of about 0.3 mg/kg, the meanmaximum brain concentration of the female mice is observed to be about11400 ng/mL at the time to maximum brain concentration of about 1.5hours and a dose of about 0.6 mg/kg, and the mean maximum brainconcentration of the female mice is observed to be about 21500 ng/mL atthe time to maximum brain concentration of about 8 hours and a dose ofabout 1 mg/kg.

Embodiment 53. A compound comprising a peptide nucleic acid sequence,wherein the peptide nucleic acid sequence comprises: (i) a series ofpeptide nucleic acid residues having a repeating triad of nucleobaseside chains; and (ii) a cell permeabilizing group attached to the seriesof peptide nucleic acid residues, wherein if the compound is subjectedto an assay, and the assay comprises:

-   -   (a) administering by intracerebroventricular administration a        dose amount of about 0.1 mg/kg to about 1.5 mg/kg of the        compound to mice;    -   (b) collecting blood samples from cava veins of the mice at a        time point between about 1 hour and 28 days post        intracerebroventricular administration;    -   (c) after the collecting the blood samples, euthanizing the mice        at a time point between about 1 hour and 28 days post        intracerebroventricular administration;    -   (d) after the euthanizing, collecting brain, intestine, liver,        lung, kidney, or muscle tissues from the mice; and    -   (e) using liquid chromatography-tandem mass spectrometry to        determine concentrations of the compound in the brain,        intestine, liver, lung, kidney, or muscle tissues that were        collected; and    -   (f) using the liquid chromatography-tandem mass spectrometry to        determine concentrations of the compound in plasma from the        blood samples that were collected from the mice,    -   then, in the assay, the compound is observed to accumulate in        the brain of the mice for at most about a month after the        administering and the compound is not observed at a detectable        level during the month in the plasma, intestine, liver, lung,        kidney, or muscle of the mice.

Embodiment 54. The compound of embodiment 53, wherein the compound isnot observed at a detectable level during the month in the plasma,intestine, liver, lung, kidney, and muscle of the mice.

Embodiment 55. The compound of embodiment 53 or embodiment 54, whereinthe compound is a gamma peptide nucleic acid.

Embodiment 56. The compound of embodiment 55, wherein the cellpermeabilizing group is an alpha substituent of the gamma peptidenucleic acid.

Embodiment 57. The compound of embodiment 55, wherein the cellpermeabilizing group is a gamma substituent of the gamma peptide nucleicacid.

Embodiment 58. The compound of embodiment 56, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are eachindependently guanidinoalkylene.

Embodiment 59. The compound of embodiment 56, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are each3-guanidino-prop-1-yl.

Embodiment 60. The compound of embodiment 56, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are each4-guanidino-but-1-yl.

Embodiment 61. The compound of any one of embodiments 53-60, wherein thepeptide nucleic acid sequence is (CTG)_(n), wherein n is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10.

Embodiment 62. The compound of any one of embodiments 53-61, wherein aC-terminus of the peptide nucleic acid sequence is bound by a peptidebond to a peptide.

Embodiment 63. The compound of any one of embodiments 53-61, wherein theC-terminus of the peptide nucleic acid sequence is bound by a peptidebond to an amidated lysine residue.

Embodiment 64. The compound of any one of embodiments 53-63, wherein anN-terminus of the peptide nucleic acid sequence of the compound isunsubstituted.

Embodiment 65. A compound comprising a peptide nucleic acid sequence,wherein the peptide nucleic acid sequence comprises: (i) a series ofpeptide nucleic acid residues having a repeating triad of nucleobaseside chains; and (ii) a cell permeabilizing group attached to the seriesof peptide nucleic acid residues, wherein if the compound is subjectedto a plasma protein binding assay, and the plasma protein binding assaycomprises:

-   -   (a) performing a human component of the plasma protein binding        assay, wherein the human component of the plasma protein binding        assay comprises (1) spiking single aliquots of human plasma with        a 10 mg/mL of a first solution of the compound to obtain at        least a second solution of the compound with concentrations of        about 1 μg/mL to about 50 μg/mL; (2) using ultracentrifugation        on the at least the second solution of the compound to separate        a mixture comprising the compounds that are bound to plasma        proteins; (3) using liquid chromatography-tandem mass        spectrometry to determine a plasma protein binding percentage in        the human plasma;    -   (b) performing a mouse component of the plasma protein binding        assay, wherein the mouse component of the plasma protein binding        assay differs from the human component of the plasma protein        binding assay only in that mouse plasma is used instead of the        human plasma;    -   (c) performing a dog component of the plasma protein binding        assay, wherein the dog component of the plasma protein binding        assay differs from the human component of the plasma protein        binding assay only in that dog plasma is used instead of the        human plasma;    -   (d) performing a minipig component of the plasma protein binding        assay, wherein the minipig component of the plasma protein        binding assay differs from the human component of the plasma        protein binding assay only in that minipig plasma is used        instead of the human plasma;    -   (e) performing a sheep component of the plasma protein binding        assay, wherein the sheep component of the plasma protein binding        assay differs from the human component of the plasma protein        binding assay only in that sheep plasma is used instead of the        human plasma; and    -   (f) performing a monkey component of the plasma protein binding        assay, wherein the monkey component of the plasma protein        binding assay differs from the human component of the plasma        protein binding assay only in that monkey plasma is used instead        of the human plasma,    -   then in the plasma protein binding assay, the plasma protein        binding percentage is at least about 85% in the human, mouse,        dog, minipig, sheep, or monkey.

Embodiment 66. The compound of embodiment 65, wherein in the plasmaprotein binding assay, the plasma protein binding percentage is at leastabout 85% in each of the human, mouse, dog, minipig, sheep, and monkey.

Embodiment 67. The compound of embodiment 65 or embodiment 66, whereinthe compound is a gamma peptide nucleic acid.

Embodiment 68. The compound of embodiment 67, wherein the cellpermeabilizing group is an alpha substituent of the gamma peptidenucleic acid.

Embodiment 69. The compound of embodiment 67, wherein the cellpermeabilizing group is a gamma substituent of the gamma peptide nucleicacid.

Embodiment 70. The compound of embodiment 68, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are eachindependently guanidinoalkylene.

Embodiment 71. The compound of embodiment 68, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are each3-guanidino-prop-1-yl.

Embodiment 72. The compound of embodiment 68, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are each4-guanidino-but-1-yl.

Embodiment 73. The compound of any one of embodiments 65-72, wherein thepeptide nucleic acid sequence is (CTG)_(n), wherein n is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10.

Embodiment 74. The compound of any one of embodiments 65-73, wherein aC-terminus of the peptide nucleic acid sequence is bound by a peptidebond to a peptide.

Embodiment 75. The compound of any one of embodiments 65-73, wherein theC-terminus of the peptide nucleic acid sequence is bound by a peptidebond to an amidated lysine residue.

Embodiment 76. The compound of any one of embodiments 65-75, wherein anN-terminus of the peptide nucleic acid sequence of the compound isunsubstituted.

Embodiment 77. The compound of any one of embodiments 65-76, wherein ata concentration of about 1 μg/mL, the plasma protein binding percentageis at least about 95% in the human, mouse, dog, minipig, sheep, ormonkey.

Embodiment 78. The compound of embodiment 77, wherein at theconcentration of about 1 μg/mL, the plasma protein binding percentage isat least about 95% in each of the human, mouse, dog, minipig, sheep, andmonkey.

Embodiment 79. A compound having the formula (I):

-   -   wherein:        -   each B is independently a nucleobase;        -   each R¹ is independently a side chain of a natural amino            acid, a guanidino(C₁-C₄)alkyl, or hydrogen;        -   each R² is independently a side chain of a natural amino            acid, a guanidino(C₁-C₄)alkyl, or hydrogen;        -   R⁵ is a sequence comprising at least one alpha amino acid            residue, beta amino acid residue, gamma amino acid residue,            or a combination thereof; hydrogen; or a water solubilizing            group;        -   n is an integer from 3-30; and        -   G is OH, NH₂, or

-   -   -   wherein:            -   R³ is hydrogen or an amino(C₁-C₄)alkyl;            -   R⁴ is a sequence comprising at least one alpha amino                acid residue, beta amino acid residue, gamma amino acid                residue, or a combination thereof; or hydrogen; and            -   m is 0 or 1;        -   wherein the compound comprises at least one            guanine-cytosine-thymine sequence;

    -   or a pharmaceutically-acceptable salt thereof.

Embodiment 80. The compound of embodiment 79, wherein each B isindependently guanine, thymine, or cytosine.

Embodiment 81. The compound of embodiment 79 or embodiment 80, whereinat least one R¹ is guanidino(C₁-C₄)alkyl.

Embodiment 82. The compound of any one of embodiments 79-81, wherein atleast one R² is guanidino(C₁-C₄)alkyl.

Embodiment 83. The compound of any one of embodiments 79-82, wherein atleast one R¹ is 4-guanidinobut-1-yl.

Embodiment 84. The compound of any one of embodiments 79-83, wherein atleast one R¹ is 3-guanidinoprop-1-yl.

Embodiment 85. The compound of any one of embodiments 79-84, wherein atleast one R² is 4-guanidinobut-1-yl.

Embodiment 86. The compound of any one of embodiments 79-85, wherein atleast one R² is 3-guanidinoprop-1-yl.

Embodiment 87. The compound of any one of embodiments 79-86, wherein Gis

Embodiment 88. The compound of embodiment 87, wherein R³ is4-aminobut-1-yl.

Embodiment 89. The compound of embodiment 87, wherein R³ is3-aminoprop-1-yl.

Embodiment 90. The compound of any one of embodiments 87-89, wherein mis 0.

Embodiment 91. The compound of any one of embodiments 87-89, wherein mis 1.

Embodiment 92. The compound of any one of embodiments 87-91, wherein R⁴is hydrogen.

Embodiment 93. The compound of any one of embodiments 87-91, wherein R⁴is a sequence comprising at least one alpha amino acid residue.

Embodiment 94. The compound of any one of embodiments 87-91 and 93,wherein R⁴ is

-   -   wherein        -   p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and        -   each R⁶ is independently hydrogen or an amino(C₁-C₄)alkyl.

Embodiment 95. The compound of any one of embodiments 87-91, 93, and 94,wherein R⁴ is

wherein

-   -   p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Embodiment 96. The compound of embodiment 95, wherein p is 3, 4, 5, 6,7, or 8.

Embodiment 97. The compound of embodiment 95, wherein p is 7.

Embodiment 98. The compound of any one of embodiments 79-97, wherein R⁵is hydrogen.

Embodiment 99. The compound of any one of embodiments 79-97, wherein R⁵is

-   -   wherein        -   each R⁷ is independently a side chain of a natural amino            acid; and        -   q is 0 or 1.

Embodiment 100. The compound of any one of embodiments 79-99, wherein R⁵is

Embodiment 101. The compound of any one of embodiments 79-99, wherein R⁵is

Embodiment 102. The compound of any one of embodiments 79-86, wherein R⁵is the water solubilizing group.

Embodiment 103. The compound of embodiment 102, wherein the watersolubilizing group is a multiply-positively charged region thatcomprises at least six consecutive building blocks.

Embodiment 104. The compound of embodiment 103, wherein each of theconsecutive building blocks independently comprises a side chain thatcarries a positive formal charge at neutral pH.

Embodiment 105. The compound of any one of embodiments 79-86 and102-104, wherein, when G is OH or NH₂, at least one of R¹ and R² is aside chain of a natural amino acid or a guanidino(C₁-C₄)alkyl, and R⁵ isnot hydrogen.

Embodiment 106. The compound of embodiment 79, which has the formula:

Embodiment 107. The compound of embodiment 106, wherein n is 6.

Embodiment 108. The compound of embodiment 106 or embodiment 107,wherein at least one R¹ is 4-guanidinobut-1-yl.

Embodiment 109. The compound of embodiment 106 or embodiment 107,wherein each R¹ is 4-guanidinobut-1-yl.

Embodiment 110. The compound of any one of embodiments 106-109, whereinat least one R² is hydrogen.

Embodiment 111. The compound of any one of embodiments 106-109, whereineach R² is hydrogen.

Embodiment 112. The compound of any one of embodiments 106-111, whereinR³ is 4-aminobut-1-yl.

Embodiment 113. The compound of any one of embodiments 106-111, whereinR³ is 3-aminoprop-1-yl.

Embodiment 114. The compound of any one of embodiments 106-113, whereinR⁴ is hydrogen.

Embodiment 115. The compound of any one of embodiments 106-113, whereinR⁴ is

-   -   wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Embodiment 116. The compound of embodiment 115, wherein p is 3, 4, 5, 6,7, or 8.

Embodiment 117. The compound of embodiment 115, wherein p is 7.

Embodiment 118. The compound of any one of embodiments 106-117, whereinR⁵ is hydrogen.

Embodiment 119. The compound of any one of embodiments 106-117, whereinR⁵ is

Embodiment 120. The compound of embodiment 79 or embodiment 106, whichhas the formula:

-   -   wherein:        -   each B^(1a), B^(2a) and B^(3a) is independently cytosine,            guanine, or thymine; and        -   n ¹ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Embodiment 121. The compound of embodiment 120, wherein n ¹ is 2.

Embodiment 122. The compound of embodiment 120 or embodiment 121,wherein each R¹ is 4-guanidinobut-1-yl.

Embodiment 123. The compound of embodiment 120, which has the formula:

Embodiment 124. The compound of embodiment 123, wherein B^(1a) iscytosine, B^(2a) is thymine and B^(3a) is guanine.

Embodiment 125. The compound of embodiment 123 or embodiment 124,wherein n ¹ is 2.

Embodiment 126. The compound of embodiment 79 or embodiment 123, whichhas the formula:

Embodiment 127. The compound of embodiment 79 or embodiment 106, whichhas the formula:

Embodiment 128. The compound of embodiment 127, wherein n is 6.

Embodiment 129. The compound of embodiment 127, which has the formula:

-   -   wherein:        -   each B^(1b), B^(2b) and B^(3b) is independently cytosine,            guanine or thymine; and        -   n ² is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Embodiment 130. The compound of embodiment 129, wherein n ² is 2.

Embodiment 131. The compound of embodiment 129 or embodiment 130,wherein each R¹ is 4-guanidinobut-1-yl.

Embodiment 132. The compound of embodiment 129, which has the formula:

Embodiment 133. The compound of embodiment 132, wherein B^(1b) iscytosine, B^(2b) is thymine and B^(3b) is guanine.

Embodiment 134. The compound of embodiment 132 or embodiment 133,wherein n ² is 2.

Embodiment 135. The compound of embodiment 79 or embodiment 132, whichhas the formula:

Embodiment 136. The compound of embodiment 79 or embodiment 106, whichhas the formula:

-   -   wherein        -   p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and        -   each R⁶ is independently hydrogen or an amino(C₁-C₄)alkyl.

Embodiment 137. The compound of embodiment 136, wherein n is 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20.

Embodiment 138. The compound of embodiment 136, wherein n is 19.

Embodiment 139. The compound of any one of embodiments 136-138, whereinat least one R¹ is hydrogen.

Embodiment 140. The compound of any one of embodiments 136-139, whereinn is greater than 1, and wherein every other R¹ is hydrogen.

Embodiment 141. The compound of any one of embodiments 136-140, whereinat least one R¹ is 4-guanidinobut-1-yl.

Embodiment 142. The compound of any one of embodiments 136-140, whereinn is greater than 1, and wherein every other R¹ is 4-guanidinobut-1-yl.

Embodiment 143. The compound of any one of embodiments 136-142, at leastone R² is hydrogen.

Embodiment 144. The compound of any one of embodiments 136-142, whereineach R² is hydrogen.

Embodiment 145. The compound of any one of embodiments 136-143, whereinat least one R² is 4-guanidinobut-1-yl.

Embodiment 146. The compound of any one of embodiments 136-143, whereinn is greater than 1, and wherein every other R² is 4-guanidinobut-1-yl.

Embodiment 147. The compound of any one of embodiments 136-146, whereinR³ is 4-aminobut-1-yl.

Embodiment 148. The compound of any one of embodiments 136-146, whereinR³ is 3-aminoprop-1-yl.

Embodiment 149. The compound of any one of embodiments 136-148, whereinR⁵ is hydrogen.

Embodiment 150. The compound of any one of embodiments 136-148, whereinR⁵ is

Embodiment 151. The compound of any one of embodiments 136-148, whereinR⁵ is

Embodiment 152. The compound of any one of embodiments 136-148, whereinR⁶ is 4-aminobut-1-yl.

Embodiment 153. The compound of any one of embodiments 136-148, whereinR⁶ is 3-aminoprop-1-yl.

Embodiment 154. The compound of any one of embodiments 136-148, whereinp is 7.

Embodiment 155. The compound of embodiment 115, wherein p is 3, 4, 5, 6,7, or 8.

Embodiment 156. The compound of any one of embodiments 79, 106, and 136,which has the formula:

-   -   wherein R⁷ is a side chain of a natural amino acid.

Embodiment 157. The compound of embodiment 156, wherein R⁷ is4-aminobut-1-yl.

Embodiment 158. The compound of embodiment 156, wherein R⁷ is3-aminoprop-1-yl.

Embodiment 159. The compound of embodiment 156, which has the structure

-   -   wherein:        -   each B^(1c), B^(2c), B^(3c) and B^(4c) is independently            cytosine, guanine or thymine; and        -   n ³ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Embodiment 160. The compound of embodiment 159, wherein B^(1c) isguanine.

Embodiment 161. The compound of embodiment 159 or embodiment 160,wherein B^(2c) is cytosine.

Embodiment 162. The compound of any one of embodiments 159-161, whereinB^(3c) is thymine.

Embodiment 163. The compound of any one of embodiments 159-162, whereinB^(4c) is guanine.

Embodiment 164. The compound of any one of embodiments 159-163, whereinn ³ is 6.

Embodiment 165. The compound of any one of embodiments 159-164, whereinp is 7.

Embodiment 166. The compound of any one of embodiments 159-165, whereinR³ is 4-aminobut-1-yl.

Embodiment 167. The compound of any one of embodiments 159-165, whereinR³ is 3-aminoprop-1-yl.

Embodiment 168. The compound of any one of embodiments 159-167, whereinR⁶ is 4-aminobut-1-yl.

Embodiment 169. The compound of any one of embodiments 159-167, whereinR⁶ is 3-aminoprop-1-yl.

Embodiment 170. The compound of any one of embodiments 159-169, whereinR⁷ is 4-aminobut-1-yl.

Embodiment 171. The compound of any one of embodiments 159-169, whereinR⁷ is 3-aminoprop-1-yl.

Embodiment 172. The compound of any one of embodiments 159-163, whichhas the structure

Embodiment 173. The compound of embodiment 79 or embodiment 159, whichhas the structure

Embodiment 174. The compound of embodiment 79 or embodiment 106, whichhas the formula:

-   -   wherein R⁷ is a side chain of a natural amino acid.

Embodiment 175. The compound of embodiment 174, wherein R⁷ is4-aminobut-1-yl.

Embodiment 176. The compound of embodiment 174, wherein R⁷ is3-aminoprop-1-yl.

Embodiment 177. The compound of any one of embodiments 79, 106, and 174,which has the structure

-   -   wherein:        -   X is

-   -   -   Y is

-   -   -   each B^(1d), B^(2d), B^(3d), B^(4d), B^(5d), B^(6d), B^(7d),            B^(8d), B^(9d), and B^(10d) is in dependently cytosine,            guanine or thymine;        -   n ⁴ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and        -   n ⁵ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Embodiment 178. The compound of embodiment 177, wherein B^(1d) isguanine.

Embodiment 179. The compound of embodiment 177 or embodiment 178,wherein B^(2d) is cytosine.

Embodiment 180. The compound of any one of embodiments 177-179, whereinB^(3d) is thymine.

Embodiment 181. The compound of any one of embodiments 177-180, whereinB^(4d) is guanine.

Embodiment 182. The compound of any one of embodiments 177-181, whereinB^(5d) is cytosine.

Embodiment 183. The compound of any one of embodiments 177-182, whereinB^(6d) is thymine.

Embodiment 184. The compound of any one of embodiments 177-183, whereinB^(7d) is guanine.

Embodiment 185. The compound of any one of embodiments 177-184, whereinB^(8d) is cytosine.

Embodiment 186. The compound of any one of embodiments 177-185, whereinB^(9d) is thymine.

Embodiment 187. The compound of any one of embodiments 177-186, whereinB^(10d) is guanine.

Embodiment 188. The compound of any one of embodiments 177-187, whereinn ⁴ is 3.

Embodiment 189. The compound of any one of embodiments 177-188, whereinn ⁵ is 2.

Embodiment 190. The compound of any one of embodiments 177-189, whereinat least one R¹ is 4-guanidinobut-1-yl.

Embodiment 191. The compound of any one of embodiments 177-189, whereineach R¹ is 4-guanidinobut-1-yl.

Embodiment 192. The compound of any one of embodiments 177-191, whereinR³ is 4-aminobut-1-yl.

Embodiment 193. The compound of any one of embodiments 177-191, whereinR³ is 3-aminoprop-1-yl.

Embodiment 194. The compound of any one of embodiments 177-194, whereinR⁷ is 4-aminobut-1-yl.

Embodiment 195. The compound of any one of embodiments 177-194, whereinR⁷ is 3-aminoprop-1-yl.

Embodiment 196. The compound of any one of embodiments 177-189, whichhas the structure:

-   -   wherein:        -   X¹ is

-   -   -    and        -   Y¹ is

Embodiment 197. The compound of any one of embodiments 79, 106, 174,177, and 196, which has the structure

-   -   wherein:        -   X² is

-   -   -    and

-   -   -   Y² is

Embodiment 198. The compound of any one of embodiments 79, 106, or 174,which has the structure

-   -   wherein:        -   L is

-   -   -   M is

-   -   -   each B^(1e), B^(2e), B^(3e), B^(4e), B^(5e), B^(6e), B^(7e),            B^(8e), B^(9e) and B^(10e) is independently cytosine,            guanine or thymine;        -   n ⁶ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and        -   n ⁷ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Embodiment 199. The compound of embodiment 198, wherein B^(1e) isguanine.

Embodiment 200. The compound of embodiment 198 or embodiment 199,wherein B^(2e) is cytosine.

Embodiment 201. The compound of any one of embodiments 198-200, whereinB^(3e) is thymine.

Embodiment 202. The compound of any one of embodiments 198-201, whereinB^(4e) is guanine.

Embodiment 203. The compound of any one of embodiments 198-202, whereinB^(5e) is cytosine.

Embodiment 204. The compound of any one of embodiments 198-203, whereinB^(6e) is thymine.

Embodiment 205. The compound of any one of embodiments 198-204, whereinB^(7e) is guanine.

Embodiment 206. The compound of any one of embodiments 198-205, whereinB^(8e) is cytosine.

Embodiment 207. The compound of any one of embodiments 198-206, whereinB^(9e) is thymine.

Embodiment 208. The compound of any one of embodiments 198-207, whereinB^(10e) is guanine.

Embodiment 209. The compound of any one of embodiments 198-208, whereinn ⁶ is 3.

Embodiment 210. The compound of any one of embodiments 198-209, whereinn ⁷ is 2.

Embodiment 211. The compound of any one of embodiments 198-210, whereinat least one R² is 3-guanidinoprop-1-yl.

Embodiment 212. The compound of any one of embodiments 198-210, whereineach R² is 3-guanidinoprop-1-yl.

Embodiment 213. The compound of any one of embodiments 198-210, whereinat least one R² is 4-guanidinobut-1-yl.

Embodiment 214. The compound of any one of embodiments 198-210, whereineach R² is 4-guanidinobut-1-yl.

Embodiment 215. The compound of any one of embodiments 198-214, whereinR³ is 4-aminobut-1-yl.

Embodiment 216. The compound of any one of embodiments 198-214, whereinR³ is 3-aminoprop-1-yl.

Embodiment 217. The compound of any one of embodiments 198-216, whereinR⁷ is 4-aminobut-1-yl.

Embodiment 218. The compound of any one of embodiments 198-216, whereinR⁷ is 3-aminoprop-1-yl.

Embodiment 219. The compound of embodiment 198, which has the structure

-   -   wherein:        -   L¹ is

-   -   -    and        -   M¹ is

Embodiment 220. The compound of any one of embodiments 79, 106, 174,198, and 219, which has the structure

-   -   wherein:        -   L² is

-   -   -    and        -   M² is

Embodiment 221. A method of treating a condition in a subject, themethod comprising administering to the subject atherapeutically-effective amount of the compound of any one ofembodiments 65-220.

Embodiment 222. The method of embodiment 221, wherein the subject is ahuman.

Embodiment 223. The method of embodiment 221 or embodiment 222, whereinthe condition is a neurological condition.

Embodiment 224. The method of embodiment 223, wherein the neurologicalcondition is Huntington's disease.

Embodiment 225. The method of embodiment 221 or embodiment 222, whereinthe condition is a polyglutamine disease.

Embodiment 226. The method of embodiment 225, wherein the polyglutaminedisease is Huntington's disease.

Embodiment 227. The method of embodiment 221 or embodiment 222, whereinthe condition is a central nervous system condition.

Embodiment 228. The method of embodiment 221 or embodiment 222, whereinthe condition is associated with aging.

Embodiment 229. The method of embodiment 221 or embodiment 222, whereinthe condition is associated with cognitive impairment.

Embodiment 230. The method of embodiment 221 or embodiment 222, whereinthe condition is associated with memory loss.

Embodiment 231. The method of embodiment 221 or embodiment 222, whereinthe condition is associated with deterioration of motor skills.

Embodiment 232. The method of any one of embodiments 221-231, whereinthe therapeutically-effective amount of the compound is from about 8μg/kg to about 200 μg/kg.

Embodiment 233. The method of any one of embodiments 221-232, whereinthe compound binds RNA.

Embodiment 234. The method of any one of embodiments 221-232, whereinthe compound binds DNA.

Embodiment 235. The method of any one of embodiments 221-234, whereinthe compound binds a CAG repeat sequence in a nucleic acid molecule.

Embodiment 236. The method of any one of embodiments 221-235, whereinthe administering is intravenous administration.

Embodiment 237. The method of any one of embodiments 221-235, whereinthe administering is subcutaneous administration.

Embodiment 238. The method of any one of embodiments 221-235, whereinthe administering is intracerebroventricular administration.

Embodiment 239. The method of any one of embodiments 221-235, whereinthe administering is oral administration.

Embodiment 240. The method of any one of embodiments 221-235, whereinthe administering is intrathecal administration.

1-32. (canceled)
 33. A compound comprising a peptide nucleic acidsequence, wherein the peptide nucleic acid sequence comprises: (i) aseries of peptide nucleic acid residues having a repeating triad ofnucleobase side chains; and (ii) a cell permeabilizing group attached tothe series of peptide nucleic acid residues, wherein if the compound issubjected to an assay, and the assay comprises: (a) administering byintracerebroventricular administration a dose amount of about 0.1 mg/kgto about 2 mg/kg of the compound to mice; (b) euthanizing the mice at atime point between about 1 hour and 28 days post intracerebroventricularadministration; (c) collecting brain tissues from the mice after theeuthanizing; and (d) using liquid chromatography-tandem massspectrometry to determine concentrations of the brain tissues in themice, then in the assay, a mean maximum brain concentration is observedin the mice at a time to maximum brain concentration of about 1 hour toabout 50 hours post administration and the mean maximum brainconcentration of the mice is observed to be about 3000 ng/mL to about22000 ng/mL.
 34. The compound of claim 33, wherein the compound is agamma peptide nucleic acid.
 35. The compound of claim 34, wherein thecell permeabilizing group is a gamma substituent of the gamma peptidenucleic acid.
 36. The compound of claim 35, wherein a plurality of gammasubstituents of the gamma peptide nucleic acid are each independentlyguanidinoalkylene.
 37. The compound of claim 35, wherein a plurality ofgamma substituents of the gamma peptide nucleic acid are each3-guanidino-prop-1-yl.
 38. The compound of claim 35, wherein a pluralityof gamma substituents of the gamma peptide nucleic acid are each4-guanidino-but-1-yl.
 39. The compound of claim 33, wherein the peptidenucleic acid sequence is (CTG)_(n), wherein n is 1, 2, 3, 4, 5, 6, 7, 8,9, or
 10. 40. The compound of claim 33, wherein a C-terminus of thepeptide nucleic acid sequence is bound by a peptide bond to a peptide.41. The compound of claim 33, wherein the C-terminus of the peptidenucleic acid sequence is bound by a peptide bond to an amidated lysineresidue.
 42. The compound of claim 33, wherein an N-terminus of thepeptide nucleic acid sequence of the compound is unsubstituted.
 43. Thecompound of claim 33, wherein if (1) the mice are male mice, the meanmaximum brain concentration of the male mice is observed to be about3110 ng/mL at the time to maximum brain concentration of about 1.5 hoursand the dose amount of about 0.3 mg/kg, the mean maximum brainconcentration of the male mice is observed to be about 6490 ng/mL at thetime to maximum brain concentration of about 4 hours and the dose amountof about 0.6 mg/kg, and the mean maximum brain concentration of the malemice is observed to be about 15500 ng/mL at the time to maximum brainconcentration of about 24 hours and the dose amount of about 1 mg/kg;and (2) if the mice are female mice, the mean maximum brainconcentration of the female mice is observed to be about 3930 ng/mL atthe time to maximum brain concentration of about 48 hours and a dose ofabout 0.3 mg/kg, the mean maximum brain concentration of the female miceis observed to be about 11400 ng/mL at the time to maximum brainconcentration of about 1.5 hours and a dose of about 0.6 mg/kg, and themean maximum brain concentration of the female mice is observed to beabout 21500 ng/mL at the time to maximum brain concentration of about 8hours and a dose of about 1 mg/kg.
 44. A method of treating a conditionin a subject, the method comprising administering to the subject atherapeutically-effective amount of the compound of claim
 33. 45. Themethod of claim 44, wherein the subject is a human.
 46. The method ofclaim 44, wherein the condition is a neurological condition.
 47. Themethod of claim 46, wherein the neurological condition is Huntington'sdisease.
 48. The method of claim 44, wherein the condition is apolyglutamine disease.
 49. The method of claim 44, wherein the conditionis a central nervous system condition.
 50. The method of claim 44,wherein the condition is associated with aging.
 51. The method of claim44, wherein the condition is associated with cognitive impairment. 52.The method of claim 44, wherein the condition is associated with memoryloss.
 53. The method of claim 44, wherein the condition is associatedwith deterioration of motor skills.
 54. The method of claim 44, whereinthe therapeutically-effective amount of the compound is from about 8μg/kg to about 200 μg/kg.
 55. The method of claim 44, wherein thecompound binds RNA.
 56. The method of claim 44, wherein the compoundbinds DNA.
 57. The method of claim 44, wherein the compound binds a CAGrepeat sequence in a nucleic acid molecule.
 58. The method of claim 44,wherein the administering is intravenous administration.
 59. The methodof claim 44, wherein the administering is subcutaneous administration.60. The method of claim 44, wherein the administering isintracerebroventricular administration.
 61. The method of claim 44,wherein the administering is oral administration. 62-260. (canceled)